Method for detecting protein-specific glycosylation

ABSTRACT

Methods are provided for detecting a glycosylated target protein in a sample. Aspects of the methods include: (a) contacting a sample comprising a probe-labeled glycosylated target protein with: (i) a first conjugate comprising a first nucleic acid tag linked to a first capture agent that specifically binds the target protein; (ii) a second conjugate comprising a second nucleic acid tag linked to a second capture agent that specifically binds the probe; and (iii) a bridging nucleic acid that hybridizes to the first and second nucleic acid tags; under conditions sufficient to specifically bind the first and second capture agents to the probe-labeled target protein and to hybridize the bridging nucleic acid to the first and second nucleic acid tags to produce a nucleic acid complex; and (b) detecting the nucleic acid complex. Also provided are compositions and kits useful in practicing various embodiments of the subject methods.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 62/189,630, filed Jul. 7, 2015, which application isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. GM059907awarded by the National Institutes of Health. The government has certainrights in the invention.

INTRODUCTION

The reversible attachment of N-acetylglucosamine (GlcNAc) to serine orthreonine side chains of intracellular proteins is a post-translationalmodification (PTM) termed O-GlcNAc. The O-GlcNAc modification regulatesdiverse cellular activities. O-GlcNAc is installed by a single enzyme,O-GlcNAc transferase (OGT), and removed by O-GlcNAc-ase (OGA). Thismodification is widespread—more than 3000 O-GlcNAc sites have beendiscovered in eukaryotic proteomes—and mediates cellular activities byregulating protein trafficking, conformational change, and byantagonizing phosphorylation. Many human pathologies exhibit aberrantO-GlcNAcylation of specific proteins. For example, hyper-glycosylationleads to altered enzymatic activity of phosphofructokinase in aggressivebreast cancers, glycogen synthase in diabetes, and CaMKII incardiovascular disease. For example, hypo-glycosylation of the tauprotein leads to an Alzheimer's disease-like state in a mouse model.O-GlcNAc also regulates pluripotency and reprogramming in stem cellsthrough the modification of numerous transcription factors. Recently,the O-GlcNAcylation of master pluripotency regulator OCT4 increasedcells' ability to maintain pluripotency. Due to its central role inregulating cellular behavior, it is thus valuable to profileO-GlcNAcylation at a proteomic level to elucidate function. As such,methods of detecting protein specific glycosylation are of interest.

SUMMARY

Methods are provided for detecting a glycosylated target protein in asample. Aspects of the methods include: (a) contacting a samplecomprising a probe-labeled glycosylated target protein with: (i) a firstconjugate comprising a first nucleic acid tag linked to a first captureagent that specifically binds the target protein; (ii) a secondconjugate comprising a second nucleic acid tag linked to a secondcapture agent that specifically binds the probe; and (iii) a bridgingnucleic acid that hybridizes to the first and second nucleic acid tags;under conditions sufficient to specifically bind the first and secondcapture agents to the probe-labeled target protein and to hybridize thebridging nucleic acid to the first and second nucleic acid tags toproduce a target protein-bound nucleic acid complex; and (b) detectingthe target protein-bound nucleic acid complex. Also provided arecompositions and kits useful in practicing various embodiments of thesubject methods.

The present disclosure provides a method for detecting a glycosylatedtarget protein in a sample, the method comprising: (a) contacting asample comprising a probe-labeled glycosylated target protein with: (i)a first conjugate comprising a first nucleic acid tag linked to a firstcapture agent that specifically binds the target protein; (ii) a secondconjugate comprising a second nucleic acid tag linked to a secondcapture agent that specifically binds the probe; and (iii) a bridgingnucleic acid that hybridizes to the first and second nucleic acid tags;under conditions sufficient to specifically bind the first and secondcapture agents to the probe-labeled target protein and to hybridize thebridging nucleic acid to the first and second nucleic acid tags toproduce a target protein-bound nucleic acid complex; and (b) detectingthe target protein-bound nucleic acid complex. The present disclosureprovides a method for detecting a glycosylated target protein in asample, the method comprising: (a) contacting a sample comprising aprobe-labeled glycosylated target protein with: (i) a first conjugatecomprising a first nucleic acid tag linked to a first capture agent thatspecifically binds the target protein; (ii) a second conjugatecomprising a second nucleic acid tag linked to a second capture agentthat specifically binds the probe; and (iii) a bridging nucleic acidthat hybridizes to the first and second nucleic acid tags; underconditions sufficient to specifically bind the first and second captureagents to the probe-labeled target protein and to hybridize the bridgingnucleic acid to the first and second nucleic acid tags to produce aglycosylated target protein-bound nucleic acid complex; and (b)detecting the glycosylated target protein-bound nucleic acid complex. Insome cases, the target protein-bound nucleic acid complex (e.g., theglycosylated target protein-bound nucleic acid complex) comprises anamplicon and the detecting comprises: amplifying the amplicon togenerate an amplification product; and detecting the amplificationproduct to provide for detection of the glycosylated target protein. Insome cases, the bridging nucleic acid comprises a first regioncomplementary to the first nucleic acid tag and a second regioncomplementary to the second nucleic acid tag. In some cases, the methodfurther comprises, prior to step (a), contacting a sample comprising ametabolically tagged glycosylated protein with a reactive probe toproduce the probe-labeled glycosylated target protein. In some cases,the sample is obtained from a eukaryotic cell comprising themetabolically tagged glycosylated protein. In some cases, the methodfurther comprises contacting the eukaryotic cell with a tagged sugarunder conditions sufficient to produce the metabolically taggedglycosylated protein. In some cases, the metabolically tagged proteincomprises a first chemoselective tag. In some cases, the firstchemoselective tag is an azide. In some cases, the reactive probecomprises a second chemoselective tag selected from the group consistingof an alkyne, an azide, a phosphine, a thiol, a maleimide or iodoacetyl,an aldehyde, an alkoxyamine. In some cases, the second chemoselectivetag is an alkyne. In some cases, the first capture agent and the secondcapture agent are independently selected from a nucleic acid, a protein,a peptide, or a small molecule. In some cases, the first capture agentis an antibody. In some cases, the second capture agent is an antibody.In some cases, the first capture agent and the second capture agents areantibodies. In some cases, the method further comprises determining theamount of total target protein in the sample. In some cases, determiningthe amount of total target protein is carried out using aproximity-based ligation assay comprising: (a) contacting the samplewith: (i) a third conjugate comprising a third nucleic acid tag linkedto a third capture agent that specifically binds a first epitope in thetarget protein; (ii) a fourth conjugate comprising a fourth nucleic acidtag linked to a fourth capture agent that specifically a second epitopein the target protein; and (iii) a bridging nucleic acid that hybridizesto the third and fourth nucleic acid tags; under conditions sufficientto specifically bind the third and fourth capture agents to theprobe-labeled target protein and to hybridize the bridging nucleic acidto the third and fourth nucleic acid tags to produce a total targetprotein-bound nucleic acid complex; and (b) detecting the targetprotein-bound nucleic acid complex. In some cases, the method comprisescomparing the level of glycosylated target protein to the level of totaltarget protein.

The present disclosure provides a composition comprising: (a) a firstconjugate comprising a first nucleic acid tag linked to a first captureagent that is capable of specifically binding a target protein; and (b)a second conjugate comprising a second nucleic acid tag linked to asecond capture agent that is capable of specifically binding a probe. Insome cases, the composition further comprises: (c) a bridging nucleicacid that is complementary to the first and second nucleic acid tags. Insome cases, the first capture agent and the second capture agent areindependently selected from a nucleic acid, a protein, a peptide, or asmall molecule. In some cases, the first capture agent is an antibody.In some cases, the second capture agent is an antibody. In some cases,the first capture agent is an antibody and the second capture agent isan antibody. In some cases, the first capture agent is an anti-targetprotein antibody and the second capture agent is an anti-biotin antibodyor an avidin moiety. In some cases, the composition further comprises aprobe-labeled glycosylated target protein.

The present disclosure provides a kit comprising: a first conjugatecomprising a first nucleic acid tag linked to a first capture agent thatis capable of specifically binding a target protein; and a secondconjugate comprising a second nucleic acid tag linked to a secondcapture agent that is capable of specifically binding a probe. In somecases, the kit further comprises a bridging nucleic acid that iscomplementary to the first and second nucleic acid tags. In some cases,the first capture agent is an antibody. In some cases, the secondcapture agent is an antibody. In some cases, the first capture agent isan antibody and the second capture agent is an antibody. In some cases,the first capture agent is an anti-target protein antibody and thesecond capture agent is an anti-biotin antibody or an avidin moiety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the reversible attachment of O-GlcNAc on Ser and Thrresidues controlled by two conserved enzymes: O-GlcNAc transferase (OGT)and O-GlcNAc-ase (OGA).

FIG. 2 shows a scheme depicting the “Click-it” method for appendingbiotin onto O-GlcNAc.

FIG. 3 shows a scheme depicting an exemplary workflow of the subjectmethods (in some cases, termed Glyco-Seq): (a) Biotin is appended ontoO-GlcNAc using the “Click-it” method; (b) Proteins are incubated withantibody-DNA conjugates targeted to both biotin and the protein ofinterest; (c) Treatment with a short strand of DNA that is complementaryto both single-stranded DNAs, and subsequent ligation allows for (d)detection of the resultant duplex DNA by standard qPCR methods.

FIG. 4 illustrates the synthesis of antibody-DNA conjugates viasuccinimidyl 4-[N-maleimidomethyl]-cyclohexane-1 carboxylate (SMCC)crosslinking.

FIG. 5A-5B show the results of detection of O-GlcNAc in a complexsample. Alpha-crystallin (Ac) was treated with either OGA or heat-killedOGA, and then added the Ac into cell lysate at 1% wt, and detectedeither for O-GlcNAc (A) or total protein level (B) using Glyco-seq. (A)OGA treated sample shows a significantly weak signal due to the loss ofO-GlcNAc. (B) Both samples showed strong signal for total protein level.This result demonstrates that the observed signal difference in (A) wasdue to differential O-GlcNAc levels. (ΔCT: change in cycle threshold; aconventional means of reporting qPCR signal relative to a controlsample).

FIG. 6 shows a graphic comparison of Glyco-seq versus Western blot.Glyco-seq signal is reported as ΔCT as described in FIG. 5A-5B. Westernblotting was performed using streptavidin-HRP.

FIG. 7 depicts an exemplary workflow for a multiplexed Glyco-seq methodto detect O-GlcNAcylation of transcription factors: (A) Mix “Click-it”labeled sample with proximity probes; (B) Ligation of DNA segments thatare in close proximity via a universal connector and ligase; (C)Amplification of target specific amplicons by addition samples from (B)into 96-well primer plates; and (D) Quantification of the amplifiedproduct with real-time qPCR and analyze the signals.

DEFINITIONS

Before describing exemplary embodiments in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used in the description.

As used herein, the term “sample” relates to a material or mixture ofmaterials, in some cases in liquid form, containing or suspected ofcontaining one or more glycosylated proteins of interest. In someembodiments, the term refers to any plant, animal, fungal, or bacterial(or other microorganism) material containing cells, cellularmetabolites, biomarkers, or other analytes of interest, such as, forexample, tissue or fluid isolated from an individual (including withoutlimitation plasma, serum, urine, cerebrospinal fluid, lymph, tears,saliva and tissue sections) or from in vitro cell culture constituents,as well as samples from the environment. A sample as described hereinmay or may not contain cells or cellular material. The term “sample” mayalso refer to a “biological sample”. As used herein, the term“biological sample” refers to a whole organism or a subset of itstissues, cells or component parts (e.g., body fluids, including, but notlimited to, blood, mucus, lymphatic fluid, synovial fluid, cerebrospinalfluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginalfluid, semen, tears, serum, plasma, feces, swabs such as those obtainedfrom the mouth, throat, nose, ears, wounds, or ulcers, tissue biopsiessuch as those obtained from tumors, organs or other body parts, ortissue sections such as those obtained from cadavers, skin, or hair).

A “biological sample” can also refer to a homogenate, lysate or extractprepared from a whole organism or a subset of its tissues, cells orcomponent parts, or a fraction or portion thereof, including but notlimited to, plasma, serum, spinal fluid, lymph fluid, the externalsections of the skin, respiratory, intestinal, and genitourinary tracts,tears, saliva, milk, blood cells, tumors and organs. In certainembodiments, the sample has been removed from an animal or plant.Biological samples may include cells. The term “cells” is used in itsconventional sense to refer to the basic structural unit of livingorganisms, both eukaryotic and prokaryotic, having at least a cellmembrane. In certain embodiments, cells include prokaryotic cells, suchas from bacteria. In other embodiments, cells include eukaryotic cells,such as cells obtained from biological samples from animals, plants orfungi. Biological samples may include pathogens such as viruses. In someembodiments, the sample is a biological sample susceptible to infectionby a pathogen, such as a virus.

As referred to herein, the term “eukaryotic cell” is used in itsconventional sense to refer to one or more cells obtained frommulti-cell organisms such animals, plants, fungi and yeast. As such,eukaryotic cells may include, but are not limited to, those obtainedfrom yeast, fungi, plants, and animals including humans and otherprimates, including non-human primates such as chimpanzees and otherapes and monkey species; farm animals such as cattle, sheep, pigs, goatsand horses; domestic mammals such as dogs and cats; laboratory animalsincluding rodents such as mice, rats and guinea pigs; birds, includingdomestic, wild and game birds such as chickens, turkeys and othergallinaceous birds, ducks, geese, and the like. In certain embodiments,eukaryotic cells include those obtained from a human being.

As used herein, the terms “determining,” “measuring,” “assessing,” and“assaying” are used interchangeably and include both quantitative andqualitative determinations.

As used herein, the terms “affinity” and “avidity” have the same meaningand may be used interchangeably herein. “Affinity” refers to thestrength of binding, increased binding affinity being correlated with alower K_(D).

Components of interest in a sample (e.g., glycosylated proteins ofinterest) are in some cases termed “sample analytes” herein. In someembodiments, the sample is a complex sample containing at least 10²,5×10², 10³, 5×10³, 10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶, 10⁷, 5×10⁷, 10⁸,10⁹10¹⁰, 10¹¹, 10¹² or more species of analyte. In certain embodiments,the sample is a sample containing 100 or fewer analytes, such as 50 orfewer, 20 or fewer, 10 or fewer, 5 or fewer, or even one analyte.

A “biopolymer” is a polymer of one or more types of repeating units,regardless of the source. Biopolymers may be found in biological systemsand may include polypeptides, polynucleotides, sugars, carbohydrates,and analogs thereof.

As used herein, the term “polypeptide” refers to a polymeric form ofamino acids of any length, including peptides that range from 2-50 aminoacids in length and polypeptides that are greater than 50 amino acids inlength. The terms “polypeptide” and “protein” are used interchangeablyherein. The term “polypeptide” includes polymers of coded and non-codedamino acids, chemically or biochemically modified or derivatized aminoacids, and polypeptides having modified peptide backbones in which theconventional backbone has been replaced with non-naturally occurring orsynthetic backbones. A polypeptide may be of any convenient length,e.g., 2 or more amino acids, such as 4 or more amino acids, 10 or moreamino acids, 20 or more amino acids, 50 or more amino acids, 100 or moreamino acids, 300 or more amino acids, such as up to 500 or 1000 or moreamino acids. “Peptides” may be 2 or more amino acids, such as 4 or moreamino acids, 10 or more amino acids, 20 or more amino acids, such as upto 50 amino acids. In some embodiments, peptides are between 5 and 30amino acids in length. The term “polypeptide” includes fusion proteins,including, but not limited to, fusion proteins with a heterologous aminoacid sequence, fusions with heterologous and native leader sequences,with or without N-terminal methionine residues; immunologically taggedproteins; fusion proteins with detectable fusion partners, e.g., fusionproteins including as a fusion partner a fluorescent protein,β-galactosidase, luciferase, etc.; and the like. In some cases, aprotein may be composed of two or more peptides and/or polypeptides.

As used herein the term “isolated,” refers to a moiety of interest thatis at least 60% free, at least 75% free, at least 90% free, at least 95%free, at least 98% free, and even at least 99% free from othercomponents with which the moiety is associated with prior topurification.

The terms “nucleic acid,” “nucleic acid molecule”, “oligonucleotide” and“polynucleotide” are used interchangeably and refer to a polymeric formof nucleotides of any length, either deoxyribonucleotides orribonucleotides, or compounds produced synthetically which can hybridizewith naturally occurring nucleic acids in a sequence specific mannersimilar to that of two naturally occurring nucleic acids, e.g., canparticipate in Watson-Crick base pairing interactions. Polynucleotidesmay have any three-dimensional structure, and may perform any function,known or unknown. Non-limiting examples of polynucleotides include agene, a gene fragment, exons, introns, messenger RNA (mRNA), transferRNA, ribosomal RNA, cDNA, recombinant polynucleotides, plasmids,vectors, isolated DNA of any sequence, control regions, isolated RNA ofany sequence, nucleic acid probes, primers and any convenient syntheticnucleic acid sequence. The term “polynucleotide” is also meant toencompass nucleic acid analogs, and mixtures of analogs and naturallyoccurring nucleic acids. Any kind of nucleic acid, such as DNA and RNA,capable of sequence specific hybridization through formation of basepairs—or similar interactions between two moieties—may be utilized toimplement the methods described herein, including artificial andunnatural nucleic acid analogs such as protein nucleic acid (PNA),locked nucleic acid (LNA), mannose nucleic acid (MNA), arabinonucleicacid (ANA), α-L-threofuranosyl-(3′→2′) nucleic acid (TNA), cyclohexenenucleic acid (CeNA), 2′-fluoroarabinose nucleic acids (FNA), glycolnucleic acid (GNA), xeno nucleic acid (XNA),2′,3′-dideoxy-1′,5′-anhydro-D-arabino-hexitol nucleic acid (HNA),intercalating nucleic acid (INA), bridged nucleic acid (BNA), andbicyclo-DNA. Sequence specific pairing of polynucleotides of interestthat find use in the subject methods may involve natural Watson-Crickbase pairing, Hoogsteen pairing, metal ion pairing, or otherconfigurations or pairings between base moieties forming hydrogen bonds,metal ion interactions, or other types of moieties forming sequencespecific pairing interactions such as unnatural base pairs (UBP) thatmay involve hydrogen bonds, hydrophobic interactions or other types ofnon-covalent bonds.

Specific pairing interactions of polynucleotides may involve natural,unnatural, artificial or modified bases. Analogs or moieties of interestinclude, but are not limited to, adenine, guanine, thymidine, cytosine,uridine, inosine, thiouridine, 5-bromouracil, methylated bases,5-methylcytocine and 5-hydroxymethylcytocine, diaminopurine,diaminopyridine, isoguanine, isocytosine, 2′-deoxyinosine,2-aminoadenine, xanthine, beta-d-glucopyranosyloxymethyluracil, d5SICS,dNaM, 2-amino-8-(2-thienyl)purine, pyridine-2-one,7-(2-thienyl)imidazo[4,5-b]pyridine, pyrrole-2-carbaldehyde,4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole,2,4-difluorotoluene, 4-methylbenzimidazole, isoquinoline,pyrrolo[2,3-b]pyridine, 2,6-bis(ethylthiomethyl)pyridine,pyridine-2,6-dicarboxamide, and mondentate pyridine.

Nucleic acid analogs of interest may include any convenient combinationof backbones, bases (or analogs thereof), and pairing moieties thatresult in a molecule capable of sequence specific binding with acomplementary nucleic acid analog of the same or different type whichcontains a complementary sequence in at least a portion of its sequence.

The term “sequence” may refer to a particular sequence of bases and/ormay also refer to a polynucleotide having a particular sequence ofbases. Thus a sequence may be information or may refer to a molecularentity, as indicated by the context of the usage.

The term “moiety” is used to refer to a portion of an entity ormolecule, in some cases having a particular function, structure, orstructural feature.

The terms “detectable moiety”, “detectable tag” and “measureable moiety”are used interchangeably herein to refer to a tag, moiety, and/ormolecule which has properties that can be detected and/or measured,directly or indirectly.

The terms “antibody,” “immunoglobulin” and their plural referentsinclude antibodies or immunoglobulins of any isotype, fragments ofantibodies which retain specific binding to antigen, including, but notlimited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies,humanized antibodies, single-chain antibodies, and fusion proteinsincluding an antigen-binding portion of an antibody and a non-antibodyprotein. The antibodies may be bound to an entity that enables theirdetection, e.g., a radioisotope, an enzyme which generates a detectableproduct, a fluorescent protein, and the like. The antibodies may befurther covalently or non-covalently conjugated to other moieties, suchas members of specific binding pairs, e.g., biotin (member ofbiotin-avidin/streptavidin specific binding pair), and the like. Theantibodies may also be bound to a solid support, including, but notlimited to, polystyrene plates or beads, and the like. Also encompassedby the terms are Fab′, Fv, F(ab′)2, and or other antibody fragments thatretain specific binding to antigen. Antibodies may exist in a variety ofother forms including, for example, Fv, Fab, and (Fab′)2, as well asbi-functional (i.e. bi-specific) hybrid antibodies (e.g., Lanzavecchiaet al., Eur. J. Immunol. 17, 105 (1987)) and in single chains (e.g.,Huston et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883 (1988); Bird etal., Science, 242, 423-426 (1988); see Hood et al., Immunology,Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller and Hood, Nature, 323,15-16 (1986)).

The terms “capable of hybridizing,” “hybridizing”, and “hybridization”as used herein refers to binding between complementary or partiallycomplementary molecules, for example as between the sense and anti-sensestrands of double-stranded DNA. Such binding is commonly non-covalentbinding, and is specific enough such that binding may be used todifferentiate between highly complementary molecules and others lesscomplementary. Examples of highly complementary molecules includecomplementary oligonucleotides, DNA, RNA, and the like, which include aregion of nucleotides arranged in the nucleotide sequence that isexactly complementary to a second nucleic acid sequence; examples ofless complementary oligonucleotides include ones with nucleotidesequences including one or more nucleotides not in the sequence exactlycomplementary to a second oligonucleotide.

The term “complementary” references a property of specific bindingbetween pairs of specific binding moieties. Specific binding moietiesare complementary if they specifically bind to each other. A pair ofspecific binding moieties that are each polynucleotides (includingnaturally occurring nucleic acids and nucleic acid analogs) may becomplementary based on their sequence complementarity. In some cases,polynucleotides are complementary if they bind to each other in ahybridization assay under stringent conditions. Portions ofpolynucleotides are complementary to each other if they followconventional base-pairing rules, e.g. A pairs with T (or U) and G pairswith C, or if they follow any convenient sequence specific pairinginteractions such as unnatural base pairs (UBP) that may involvehydrogen bonds, hydrophobic interactions or other types of non-covalentbonds. “Complementary” includes embodiments in which there is anabsolute sequence complementarity, and also embodiments in which thereis a substantial sequence complementarity. Additional examples ofspecific binding pairs which may be considered complementary includeantibody-antigen binding pairs, receptor-ligand binding pairs, nucleicacid aptamer-protein binding pairs and the like.

“Absolute sequence complementarity” means that there is 100% sequencecomplementarity between a first polynucleotide and a secondpolynucleotide, i.e. there are no insertions, deletions, orsubstitutions in either of the first and second polynucleotides withrespect to the other polynucleotide (over the complementary region). Putanother way, every base (or analog thereof) of the complementary regionis paired with its complementary base (or analog thereof) bybase-pairing or other specific pairing as described herein.

“Substantial sequence complementarity” permits one or more relativelysmall (in some cases, less than 10 bases, e.g. less than 5 bases,typically less than 3 bases, more typically a single base) insertions,deletions, or substitutions in the first and or second polynucleotide(over the complementary region) relative to the other polynucleotide.The complementary region is the region that is complementary between afirst polynucleotide and a second polynucleotide (e.g. a distinctsequence of a nucleic acid target molecule and a nucleic acid captureagent). Complementary sequences are in some cases embedded within largerpolynucleotides, thus two relatively long polynucleotides may becomplementary over only a portion of their total length. Thecomplementary region may be of any convenient length, and is in somecases at least 5 bases long, such as at least 7 bases long, at least 12bases long, at least 15 bases long, at least 20 bases long, at least 25bases long, at least 30 bases long, at least 40 bases long, at least 50bases long, at least 60 bases long, at least 70 bases long, at least 80bases long, at least 90 bases long, at least 100 bases long, at least200 bases long, at least 300 bases long, at least 400 bases long, atleast 500 bases long, at least 600 bases long, at least 700 bases long,at least 800 bases long, at least 1000 bases long, at least 2000 baseslong, at least 3000 bases long, at least 4000 bases long, at least 5000bases long, or even longer.

The terms “hybridizing specifically to,” “specific hybridization,”“selectively hybridize to,” and the like are used herein to refer to thebinding, duplexing, or hybridizing of a nucleic acid moleculepreferentially to a particular nucleotide sequence under “stringentconditions.”

The term “stringent conditions” refers to conditions under which a firstmolecule, e.g., a first nucleic acid, will bind preferentially to asecond molecule, e.g., a second nucleic acid, and to a lesser extent to,or not at all to, e.g., other sequences. Put another way, the term“stringent hybridization conditions” as used herein refers to conditionsthat are compatible to produce complexes (e.g., duplexes) betweencomplementary binding members, e.g., between a sequence of a nucleicacid capture agent and a complementary sequence of a target nucleicacid. In some instances, the first and second complementary bindingmembers include molecules selected from a protein, such an antibody,which specifically binds to a complementary antigen and not to othermolecules under stringent conditions. Stringent conditions for specificbinding involving biomolecules such as proteins may include high saltconcentrations and high temperatures.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization are sequencedependent, and are different under different environmental parameters.Stringent hybridization conditions can include, e.g., hybridization in abuffer including 50% formamide, 5× saline sodium citrate (SSC), and 1%sodium dodecyl sulfate (SDS) at 42° C., or hybridization in a bufferincluding 5×SSC and 1% SDS at 65° C., both with a wash of 0.2×SSC and0.1% SDS at 65° C. Exemplary stringent hybridization conditions can alsoinclude a hybridization in a buffer of 40% formamide, 1 M NaCl, and 1%SDS at 37° C., and a wash in 1×SSC at 45° C. Yet additional stringenthybridization conditions include hybridization at 60° C. or higher and3×SSC (450 mM NaCl/45 mM sodium citrate) or incubation at 42° C. in asolution containing 30% formamide, 1M NaCl, 0.5% sodium sarcosine, 50 mM2-(N-morpholino)ethanesulfonic acid, pH 6.5. Those of ordinary skillwill readily recognize that alternative but comparable hybridization andwash conditions can be utilized to provide conditions of similarstringency.

In certain embodiments, the stringency of the wash conditions may affectthe degree to which nucleic acid molecules specifically hybridize.Suitable wash conditions may include, e.g.: a salt concentration ofabout 0.02 M at pH 7 and a temperature of at least about 50° C. or about55° C. to about 60° C.; or, a salt concentration of about 0.15 M NaCl at72° C. for about 15 min; or, a salt concentration of about 0.2×SSC at atemperature of at least about 50° C. or about 55° C. to about 60° C. forabout 1 to about 20 min; or, multiple washes with a solution with a saltconcentration of about 0.1×SSC containing 0.1% SDS at 20 to 50° C. for 1to 15 min; or, equivalent conditions. Stringent conditions for washingcan also be, e.g., 0.2×SSC/0.1% SDS at 42° C. In instances wherein thenucleic acid molecules are oligodeoxynucleotides (i.e. oligonucleotidesmade up of deoxyribonucleotide subunits), stringent conditions caninclude washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-baseoligos), and 60° C. (for 23-base oligos). See Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N.Y.), for detailed descriptions of equivalenthybridization and wash conditions and for reagents and buffers, e.g.,SSC buffers and equivalent reagents and conditions.

Stringent hybridization conditions may also include a “prehybridization”of aqueous phase nucleic acids with complexity-reducing nucleic acids tosuppress repetitive sequences. For example, certain stringenthybridization conditions include, prior to any hybridization tosurface-bound polynucleotides, hybridization with random sequencesynthetic oligonucleotides (e.g. 25-mers), or the like. Other stringenthybridization conditions are known in the art and may also be employed,as appropriate.

The term “amplicon” as used herein refers to a nucleic acid complex thatis the source of an amplified nucleic acid or the initiating nucleicacid in a nucleic acid amplification reaction. A “nucleic acid complex”refers to two or more joined nucleic acids including but not limited toe.g., a duplex, a triplex, a quadruplex, a pentaplex, a hexaplex, andthe like. The nucleic acids of a nucleic acid complex may be joined,e.g., hybridized, through hydrogen bonding interactions includingWatson-Crick base-pairing. In some instances, two or more nucleic acidsof a nucleic acid complex may be ligated together through the covalentlinking of two ends of individual nucleic acid molecules, e.g., throughthe use of an enzyme that catalyzes the covalent joining of nucleicacids or ligases. In an amplification reaction additional amplificationproduct may be amplified from amplification product that is the resultof the initial amplicon and, as such, the term amplicon may also referto the product of an amplification reaction which is subsequently usedin further amplification, however, as used herein, an amplicon generallyrefers to the initial polynucleotide or polynucleotide complex fromwhich amplification is initiated.

The term “ligase” as referred to herein refers collectively to enzymesthat catalyze the covalent joining of two adjacent ends of a nucleicacid molecule or molecules. For example, a nucleic acid ligase maycatalyze the formation of a phosphodiester bond between juxtaposed 5′phosphate and 3′ hydroxyl termini in single stranded or double strandednucleic acid, including, e.g., ssDNA, dsDNA, ssRNA, and dsRNA. Ligasesmay ligate nucleic acid hybridized to a complementary nucleic acid ormay ligate in the absence of a complementary nucleic acid. Anyconvenient ligase may find use in the methods described herein includingbut not limited to, e.g., naturally occurring ligases, synthetic orrecombinant ligases, mutant ligases, DNA ligases, RNA ligases,sticky-end ligases, blunt end ligases, nick-repair ligases, thermostableligases, thermolabile ligases, T4 DNA ligase, T3 DNA ligase, T7 DNAligase, E. coli DNA ligase, Taq DNA ligase, Thermococcus DNA ligase,Chlorella virus DNA Ligase, T4 RNA ligase 1, T4 RNA ligase 2,Methanobacterium thermoautotrophicum DNA/RNA ligase, and the like.

The term “primer” or “oligonucleotide primer” as used herein, refers toan oligonucleotide which acts to initiate synthesis of a complementarynucleic acid strand when placed under conditions in which synthesis of aprimer extension product is induced, e.g., in the presence ofnucleotides and a polymerization-inducing agent such as a DNA or RNApolymerase and at suitable temperature, pH, metal concentration, andsalt concentration. Primers are generally of a length compatible withtheir use in synthesis of primer extension products, and may be in therange of between 8 to 100 nucleotides in length, such as 10 to 75, 15 to60, 15 to 40, 18 to 30, 20 to 40, 21 to 50, 22 to 45, 25 to 40, and soon, including in the range of between 18-40, 20-35, 21-30 nucleotideslong, and any length between the stated ranges. In some instances,primers can be in the range of between 10-50 nucleotides long, such as15-45, 18-40, 20-30, 21-25 and so on, and any length between the statedranges. In some embodiments, the primers are usually not more than about10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,55, 60, 65, or 70 nucleotides in length.

The terms “bind” and “bound” as used herein refer to a bindinginteraction between two or more entities. Where two entities, e.g.,molecules, are bound to each other, they may be directly bound, i.e.,bound directly to one another, or they may be indirectly bound, i.e.,bound through the use of an intermediate linking moiety or entity. Ineither case the binding may be covalent; e.g., through covalent bonds;or non-covalent, e.g., through ionic bonds, hydrogen bonds,electrostatic interactions, hydrophobic interactions, Van der Waalsforces, or a combination thereof.

As used herein, the terms “chemoselective functional group” and“chemoselective tag” are used interchangeably and refer tochemoselective reactive groups that selectively react with one anotherto form a covalent bond. Chemoselective functional groups of interestinclude, but are not limited to, thiols and maleimide or iodoacetamide,as well as groups that can react with one another via Click chemistry,e.g., azide and alkyne groups (e.g., cyclooctyne groups).

The term “contacting” is used herein in its conventional sense to referto placing two or more aspects in proximity or providing an interactionor communication between two or more aspects. For example, contactingmay mean exposing (e.g., incubating with and/or allowing direct physicalcontact between) one aspect (e.g., an isotopic labeling composition) toanother aspect (a cell). Contacting may also mean, for example, allowingone aspect to integrate with and/or penetrate and/or chemically reactwith another aspect.

The methods described herein include multiple steps. Each step may beperformed after a predetermined amount of time has elapsed betweensteps, as desired. As such, the time between performing each step may be1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds ormore, 5 minutes or more, 10 minutes or more, 60 minutes or more andincluding 5 hours or more. In certain embodiments, each subsequent stepis performed immediately after completion of the previous step. In otherembodiments, a step may be performed after an incubation or waiting timeafter completion of the previous step, e.g., a few minutes to anovernight waiting time.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “atarget protein” includes a plurality of such target proteins andreference to “the target protein” includes reference to one or moretarget proteins and equivalents thereof known to those skilled in theart, and so forth. It is further noted that the claims may be drafted toexclude any optional element. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely,” “only” and the like in connection with the recitation of claimelements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

As summarized above, aspects of the present disclosure include a methodfor detecting a glycosylated target protein in a sample. Aspects of themethod include labelling a metabolically tagged glycosylated targetprotein with a reactive probe to produce a probe-labeled glycosylatedtarget protein. Detection of the probe-labeled glycosylated targetprotein may be achieved by specifically binding two conjugates to theprobe-labeled glycosylated protein: a first conjugate which specificallybinds the target protein and a second conjugate which specifically bindsthe probe. The first and second conjugates include first and secondnucleic acid tags, respectively. When the first and second conjugatesare specifically bound to the probe-labeled glycosylated target protein,the first and second nucleic acid tags are in proximity to each other.Any convenient methods of proximity ligation assays may be adapted inthe subject methods to provide for detection of the specifically boundprobe-labeled glycosylated target protein.

Aspects of the method include hybridizing a bridging nucleic acid to theproximate first and second nucleic acid tags to produce a targetprotein-bound nucleic acid complex that may be subsequently detectedusing polymerase chain reaction (PCR). The bridging nucleic acidincludes a first region complementary to the first nucleic acid tag anda second region complementary to the second nucleic acid tag. The firstconjugate includes a first capture agent that specifically binds totarget protein (e.g., glycosylated or non-glycosylated target protein).The second conjugate includes a second capture agent that specificallybinds the probe which may installed on the target protein viachemoselective labelling to a tagged sugar on a metabolically taggedprotein (e.g., target protein or non-target protein). As such, in somecases, the formation of the target protein-bound nucleic acid complexmay occur only for target proteins that are metabolically taggedglycosylated target proteins. Non-target proteins and target proteinsthat are not metabolically tagged may be easily distinguished from theglycosylated target protein using the subject methods.

FIG. 3 shows a scheme depicting an exemplary workflow of the subjectmethods (Glyco-Seq). In step (a), biotin (303) is attached onto ametabolically labelled O-GlcNAc (302) of target protein (301) using the“Click-it” method. In step (b), the biotin probe-labelled target protein(304) is incubated with first and second antibody-DNA conjugatestargeted to either biotin (306) or the target protein (305). Step (c)depicts the treatment of the specifically bound target protein 311 witha bridging nucleic acid (309), i.e., a short strand of DNA that iscomplementary to both single-stranded nucleic acid tags (307 and 308) ofthe first and second conjugates to produce a target protein-boundnucleic acid complex (310). In step (d), detection of the resultantcomplex is achieved by conventional qPCR methods.

Each of these components that find use in the subject methods andcompositions are now described in more detail, followed by furtherdetails of the methods of using the same.

Probe-Labeled Glycosylated Target Protein

Any convenient samples (e.g., as defined herein) may be analyzedaccording to the subject methods. The sample may include, or besuspected of including, one or more glycosylated target proteins ofinterest. The compositions and methods of the present disclosure may beutilized in connection with the qualitative and/or quantitativedetection of any of a wide variety of glycosylated target proteins ofinterest. As used herein, the term “a target protein” refers to allmembers of the target protein family, and fragments thereof. The targetprotein may be any protein of interest, such as a therapeutic ordiagnostic target, including but not limited to: hormones, growthfactors, receptors, enzymes, cytokines, osteoinductive factors, colonystimulating factors and immunoglobulins. The term “target protein” isintended to include recombinant and synthetic molecules, which can beprepared using any convenient recombinant expression methods or usingany convenient synthetic methods, or purchased commercially. A targetprotein may be isolated, substantially purified, or present within thenative milieu (e.g., on a cell surface or within a cell, includingwithin a host animal, e.g., a mammalian animal, such as a murine host(e.g., rat, mouse), hamster, canine, feline, bovine, swine, and thelike). Protein targets of interest include, for example, cell surfacereceptors, signal transduction factors, and hormones. Nucleic acidtargets of interest include, for example, DNA and RNA targets. Cellulartargets of interest include, for example, mammalian cells (particularlyhuman cells, e.g., human cancer cells) stem cells, and bacterial cells.

In some embodiments, the glycosylated target protein is present in vitroin a cell-free reaction. In other embodiments, the glycosylated targetprotein is present in a cell and/or displayed on the surface of a cell.In many embodiments of interest, the glycosylated target protein is in aliving cell; on the surface of a living cell; in a living organism,e.g., in a living multicellular organism. Suitable living cells includecells that are part of a living multicellular organism; cells isolatedfrom a multicellular organism; immortalized cell lines; and the like.The protein may be composed of D-amino acids, L-amino acids, or both,and may be further modified, either naturally, synthetically, orrecombinantly, to include other moieties. For example, the glycosylatedtarget polypeptide may be a lipoprotein, a glycoprotein, or other suchmodified protein.

In some embodiments, the subject method includes contacting a eukaryoticcell with a tagged sugar under conditions sufficient to produce ametabolically tagged glycosylated protein. Aspects of the method includemetabolically embedding a chemoselective tag into one or more molecules(e.g., glycans). By “metabolically embedding”, as used herein, is meantinserting an aspect (e.g., one or more chemoselective tags) into one ormore metabolic processes (e.g., metabolic processes occurring within aeukaryotic cell). In some aspects, metabolic processes are associatedwith a glycan biosynthetic pathway (e.g., the gna1Δ yeast hexosaminebiosynthetic pathway). As used herein, the term “glycan” refers to apolysaccharide or oligosaccharide.

In some cases, the target protein is tagged with an azido-sugar.Molecules comprising an azide and suitable for use in the presentinvention, as well as methods for producing azide-comprising moleculessuitable for use in the present disclosure, are well known in the art.Any convenient methods of metabolically tagging a glycosylated targetprotein may be adapted for use in the subject methods. In general, thetarget protein includes at least one azide for reaction with the secondconjugate according to the subject methods, but may comprise 2 or more,3 or more, 5 or more, 10 or more azides. The number of azides that maybe present in a target protein may vary according to the particularapplication of the reaction, the nature of the target protein itself,and other considerations which will be readily apparent to theordinarily skilled artisan in practicing the invention as disclosedherein.

The target protein can be generated in vitro and then introduced intothe cell using any of a variety of methods well known in the art (e.g.,microinjection, liposome or lipofectin-mediated delivery,electroporation, etc.), which methods will vary according to the natureof the protein to be targeted for detection and can be readily andappropriately selected by the ordinarily skilled artisan. The finaltarget protein can also be generated in vivo by exploiting a host cell'snatural biosynthetic machinery. For example, the cell can be providedwith a biocompatible azide-derivative of a substrate for synthesis ofthe desired target protein, which substrate is processed by the cell toprovide an azide-derivative of the desired final target protein. Forexample, where the target protein is a cell surface glycoprotein, thecell can be provided with an azide derivative of a sugar residue foundwithin the glycoprotein, which is subsequently processed by the cellthrough natural biosynthetic processes to produce a modifiedglycoprotein having at least one modified sugar moiety comprising anaccessible azide group.

The metabolically tagged target protein can also be produced in vivousing any convenient methods. For example, unnatural amino acids havingazides can be incorporated into recombinant polypeptides expressed in E.coli (see, e.g., Kiick et al. (2000) Tetrahedron 56:9487). Suchrecombinantly produced polypeptides can be detected in a sampleaccording to the subject methods.

In one embodiment, the target molecule is a carbohydrate-containingmolecule (e.g., a glycoprotein; a polysaccharide; etc.), and an azidegroup is introduced into the target molecule using a syntheticsubstrate. In some embodiments, the synthetic substrate is an azidederivative of a sugar utilized in production of a glycosylated molecule.In some embodiments, the synthetic substrate is an azide derivative of asugar utilized in production of a cell surface molecule, e.g., in theglycoprotein biosynthetic pathway. For example, the host cell can beprovided with a synthetic sialic acid azido-derivative, which isincorporated into the pathway for sialic acid biosynthesis, eventuallyresulting in the incorporation of the synthetic sugar residue inglycoproteins. In some embodiments, the glycoproteins are displayed onthe cell surface.

In one example, the synthetic substrate is an azido derivative ofmannosamine of the general formula:

where n is from 1 to 6, generally from 1 to 4, more usually 1 to 2, andR₁, R₂, R₃, and R₄ are independently hydrogen or acetyl. In someembodiments, the substrate is N-azidoacetylmannosamine (n=1) or anacetylated derivative thereof, or N-azidopropanoylmannosamine (n=2) oran acetylated form thereof.

In another embodiment, the synthetic substrate is an azido sugarderivative of a general formula of, for example:

either of which can be incorporated into the sialic acid biosynthesispathway, and where n is from 1 to 6, generally from 1 to 4, more usually1 to 2, and R₂, R₃, and R₄ are independently hydrogen or acetyl.

In another embodiment, the synthetic substrate is an azido sugarderivative of a general formula of, for example:

where R₁, R₂, R₃, and R₄ are independently hydrogen or acetyl, and wherethe synthetic substrate is incorporated into biosynthetic pathwaysinvolving fucose.

In another embodiment, the synthetic substrate is an azido sugarderivative of a general formula of, for example:

where n is from 1 to 6, generally from 1 to 4, more usually 1 to 2, andR₁, R₂, R₃, and R₄ are independently hydrogen or acetyl, and which isincorporated into biosynthetic pathways involving galactose.

As such, a variety of methods may be used to provide metabolicallytagged glycosylated proteins, e.g., in a sample of interest. In somecases, the metabolically tagged glycosylated proteins include ametabolic tag that is an azide.

Any convenient methods and functional groups that find use inbioorthogonal or chemoselective conjugation reactions may be adapted foruse in the subject methods to label a metabolically tagged glycosylatedprotein with a probe, e.g., via chemoselective reaction with themetabolic tag. Chemoselective functional groups of interest which mayfind use in the subject methods as either metabolic tags or in reactiveprobes which are capable of conjugation to the metabolic tags, includebut are not limited to, aldehydes, azides, nitrones, nitrile oxides,diazo compounds, tetrazines, tetrazoles, quadrocyclanes, alkenes,alkynes (e.g., strained alkynes) and iodobenzenes. Bioorthogonalligation reactions of interest include, but are not limited to, thosereactions described in Table 1 of Debets et al. “Bioorthogonal labellingof biomolecules: new functional handles and ligation methods”, Org.Biomol. Chem., 2013, 11, 6439-6455, the disclosure of which is hereinincorporated by reference. In certain embodiments, the metabolicallytagged protein includes an azide tag and may be labelled with anazide-reactive probe. Any convenient azide-reactive functional groupsmay be utilized to provide for chemoselective ligation of a reactiveprobe to a metabolically tagged protein that includes an azide tag.

The “Click-it” method of detecting O-GlcNAc in lysates may be adaptedfor use in the subject methods to attach any convenient probe (e.g., asdescribed herein) to a metabolically azide tagged and glycosylatedtarget protein. In some cases, a probe such as a biotin moiety may beattached onto O-GlcNAc for facile detection (FIG. 2). FIG. 2 depicts anexemplary scheme for metabolically tagging a glycosylated target proteinwith a tagged sugar and then subsequently labelling it via achemoselective conjugation (e.g., Click-it conjugation). First, O-GlcNAcis chemo-enzymatically modified by treatment with a permissive galactosetransferase that introduces an azide-containing monosaccharide(N-azidoacetylgalactosamine, GalNAz). The azide is then reacted with analkyne-biotin reagent and detection using anti-biotin antibodies is thenperformed. All O-GlcNAcylated proteins in a sample (e.g., a cell lysate)are simultaneously labelled (e.g., biotinylated) using the method. Thedetection of the O-GlcNAcylation state of a particular target proteinmay then be achieved via a second binding event of the target proteinfor subsequent analysis.

In some embodiments, the present disclosure provides for attachment of areactive probe to an azide-modified target protein. The methodsgenerally involve reacting an azide-modified target protein with areactive probe including a strained alkyne (e.g., a cycloalkyne) tochemoselectively label the target protein with the probe.

Strained Alkynes

Any convenient strained alkynes may find use in the subject methods tolabel a glycosylated target protein of interest with a probe. As usedherein, the term “strained alkyne” refers to an alkyne containing groupor molecule where the alkyne has increased reactivity due to an inherentsteric strain (e.g., a ring strain) on the linear alkyne group. Analkyne of interest may be strained in a variety of ways, such as theintroduction of a ring structure, or the introduction of stericrepulsion into the alkyne containing group to place mechanical stress onthe carbon-carbon triple bond which can increase its reactivity.Strained alkynes of interest include those that find use instrain-promoted azide alkyne cycloaddition reactions (SPAAC), includingazide bioconjugation reactions. In some cases, the “strained alkyne” isa cyclic alkyne, such as a cycloheptyne, a cyclooctyne, a cyclononyne,or a heterocyclic analog thereof.

A variety of strained alkynes may be adapted for incorporation into areactive strained alkyne-labeled probe for use in labelling theglycosylated target protein. As used herein, the term “reactive probe”refers to a reagent for labelling a metabolically tagged andglycosylated target protein that includes a probe moiety and achemoselective functional group compatible with the metabolic tag ofinterest. In some cases, the reactive probe is a “reactive strainedalkyne-labeled probe” that includes a strained alkyne (e.g., asdescribed herein) that is modified to include a linked probe (e.g., asdescribed herein). Any of the strained alkynes described herein may beadapted to include an optional linker for attachment to a probe ofinterest, e.g., via covalent attachment of a linker or cargo agent to ahydroxyl group or a carboxylic acid group, or derivative thereof, of thestrained alkynes described herein.

In certain embodiments, the strained alkyne is described by the formula:

In certain embodiments, the strained alkyne is described by the formula:

where X in some cases may be H or F.

In certain embodiments, the strained alkyne is described by the formula:

In certain embodiments, the strained alkyne is described by the formula:

In certain embodiments, the strained alkyne is described by the formula:

where R is H or an optional linker.

In certain embodiments, the strained alkyne is described by the formula:

-   -   where an optional linker or cargo agent may be attached at any        convenient location of the TMTH strained alkyne, such as at a        ring position alpha to the S atom.

In certain embodiments, the strained alkyne is described by the formula:

In certain embodiments, the strained alkyne is described by the formula:

In certain embodiments, the strained alkyne is described by the formula:

In certain embodiments, the strained alkyne is described by the formula:

In certain embodiments, the strained alkyne is described by the formula:

In certain embodiments, the strained alkyne is described by the formula:

In certain embodiments, the strained alkyne is described by the formula:

Cyclooctynes of interest include, but are not limited to,dibenzoazocyclooctyne (DBCO/DIBAC), a dibenzocyclooctyne (DIBO) (e.g.,DIBO1, DIBO2 or S-DIBO), a difluorocyclooctyne (DIFO) (e.g., DIFO1, 2 or3), OCT1, OCT2, OCT3, MOFO, BCN, TMTH, DIMAC, BARAC, COMBO, andfluorogenic cyclooctynes such as CoumBARAC or Fl-DIBO. In certainemodiments, the cyclooctyne is selected from: dibenzoazocyclooctyne, adibenzocyclooctyne, a difluorocyclooctyne, OCT1, OCT2, OCT3, MOFO, BCN,TMTH, DIMAC, BARAC and COMBO.

In some embodiments, the cyclooctyne is selected from:

The strained alkyne may be covalently attached to a probe of interestdirectly or indirectly via an optional linker (L) (e.g., as describedherein). Exemplary linking groups and linkages and methods of using thesame are described in e.g., Hermanson, “Bioconjugate Techniques” 2ndEdition, Academic Press, 2008. The linker may be cleavable ornon-cleavable. For instance, in certain embodiments, L includes a groupselected from alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino,substituted amino, carboxyl, carboxyl ester, acyl amino, alkylamide,substituted alkylamide, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, andsubstituted heterocyclyl. In certain embodiments, the linker (L)includes an alkyl or substituted alkyl group. In certain embodiments, Lincludes an alkenyl or substituted alkenyl group. In certainembodiments, L includes an alkynyl or substituted alkynyl group. Incertain embodiments, L includes an alkoxy or substituted alkoxy group.In certain embodiments, L includes an amino or substituted amino group.In certain embodiments, L includes a carboxyl or carboxyl ester group Incertain embodiments, L includes an acyl amino group. In certainembodiments, L includes an alkylamide or substituted alkylamide group.In certain embodiments, L includes an aryl or substituted aryl group. Incertain embodiments, L includes a heteroaryl or substituted heteroarylgroup. In certain embodiments, L includes a cycloalkyl or substitutedcycloalkyl group. In certain embodiments, L includes a heterocyclyl orsubstituted heterocyclyl group.

In certain embodiments, L includes a polymer. For example, the polymermay include a polyalkylene glycol and derivatives thereof, includingpolyethylene glycol, methoxypolyethylene glycol, polyethylene glycolhomopolymers, polypropylene glycol homopolymers, copolymers of ethyleneglycol with propylene glycol (e.g., where the homopolymers andcopolymers are unsubstituted or substituted at one end with an alkylgroup), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone,combinations thereof, and the like. In certain embodiments, the polymeris a polyalkylene glycol. In certain embodiments, the polymer is apolyethylene glycol.

Probes

Aspects of the subject methods include labelling a glycosylated targetprotein with a reactive probe. As used herein the term “probe” refers toa moiety that is capable of being recognized either directly orindirectly through a specific binding member. A reactive probe furtherincludes (in addition to the probe moiety) a chemoselective group forconjugation to the metabolic label of interest. As such, a targetprotein to which a reactive probe has been chemoselectively attached(e.g., as described herein) may be specifically recognized via thebinding of a compatible specific binding member which specifically bindsto the probe (e.g., a capture agent).

In some cases, the probe is one member of a pair of specific bindingmoieties. Any convenient specific binding member may be utilized as aprobe in the subject methods to label a target protein. In someembodiments, the probe comprises a nucleic acid segment, nucleic acidanalog segment, protein (including, for instance, an antibody, receptorprotein, or enzyme), ligand, receptor, substrate, or hapten.

The terms “specific binding,” “specifically bind,” and the like, referto the ability of a first binding molecule or moiety (e.g., atarget-specific binding moiety such as a capture agent or a firstspecific binding moiety) to preferentially bind directly to a secondbinding molecule or moiety (e.g., a target molecule or a second specificbinding moiety) relative to other molecules or moieties in a reactionmixture. In certain embodiments, the affinity between a first bindingmolecule or moiety and a second binding molecule or moiety when they arespecifically bound to each other is characterized by a K_(D)(dissociation constant) of less than 10⁻⁶ M, less than 10⁻⁷ M, less than10⁻⁸ M, less than 10⁻⁹ M, less than 10⁻¹⁰ M, less than 10⁻¹¹ M, lessthan 10⁻¹² M, less than 10⁻¹³ M, less than 10⁻¹⁴ M, or less than 10⁻¹⁵M. In some cases, the affinity between a capture agent and analyte whenthey are specifically bound in a capture agent/analyte complex is atleast 10⁻⁸ M, at least 10⁻⁹ M, or at least 10⁻¹⁰ M. In some instances, aspecific binding interaction will discriminate between desirable andundesirable analytes in a sample with a specificity of 10-fold or morefor a desirable analyte over an undesirable analytes, such as 100-foldor more, or 1000-fold or more.

As used herein, a “member of a specific binding pair” is a member of apair of molecules or entities that takes part in a specific bindinginteraction. Where a first member of the specific binding pair isidentified, the identity of the second member of the specific bindingpair may be readily identifiable. It should be noted that when eithermember of the binding pair is referred to as the first member, theremaining member is understood to be the second member and vice versa.Examples of specific binding pair interactions include immuneinteractions such as antigen/antibody and hapten/antibody as well asnon-immune interactions such as complementary nucleic acid binding,complementary protein-protein interactions, a sugar and a lectinspecific therefore, an enzyme and an inhibitor therefore, an apoenzymeand cofactor, a hormone and a receptor therefore, biotin/avidin andbiotin/streptavidin.

As used herein, the term “biotin moiety” refers to an affinity agentthat includes biotin or a biotin analogue such as desthiobiotin,oxybiotin, 2′-iminobiotin, diaminobiotin, biotin sulfoxide, biocytin,etc. Biotin moieties bind to streptavidin with an affinity of at least10⁻⁸M. A biotin affinity agent may also include a linker, e.g.,-LC-biotin, -LC-LC-Biotin, -SLC-Biotin or -PEG_(n)-Biotin where n is3-12.

Capture Agents

Aspects of the subject methods include contacting the sample with: afirst conjugate including a first nucleic acid tag linked to a firstcapture agent that specifically binds a target protein; and a secondconjugate including a second nucleic acid tag linked to a second captureagent that specifically binds a probe, to form a complex.

As used herein the terms “affinity agent” and “capture agent” are usedinterchangeably and refer to an agent that binds a target moiety (e.g.,protein or probe) through an interaction that is sufficient to permitthe agent to extract the target moiety of interest from a mixture ofdifferent analytes and/or other sample components. The bindinginteraction may be mediated by an affinity region of the capture agent.Capture agents may “specifically bind” to one or more target moieties.Thus, the term “capture agent” refers to a molecule or a multi-molecularcomplex which can specifically bind a target moiety, e.g., specificallybind a target protein or probe for the capture agent with a dissociationconstant (K_(D)) of 10⁻⁶ or less without binding to other targetmoieties, such as 10⁻⁶ M or less, 10⁻⁷ M or less, including 10⁻⁸ M orless, e.g., 10⁻⁹ M or less, 10⁻¹⁰ M or less, 10⁻¹¹ M or less, 10⁻¹² M orless, 10⁻¹³ M or less, 10⁻¹⁴ M or less, 10⁻¹⁵ M or less, 10⁻¹⁶ M orless, 10⁻¹⁷ M or less, 10⁻¹⁸ M or less, or even less.

The term “capture agent/target complex” refers to a complex that resultsfrom the specific binding of a capture agent with a target moiety (e.g.,a target protein or a probe). The complex may be part of a largercomplex (e.g., a sandwich complex). A capture agent and a target moietyfor the capture agent will typically specifically bind to each otherunder “conditions suitable for specific binding”, where such conditionsare those conditions (in terms of salt concentration, pH, detergent,protein concentration, temperature, etc.) which allow for binding tooccur between capture agents and analytes in solution. Such conditions,for example with respect to antibodies and their antigens, are wellknown in the art (see, e.g., Harlow and Lane (Antibodies: A LaboratoryManual Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)).Conditions suitable for specific binding in some cases permit captureagents and target pairs that have a dissociation constant (K_(D)) ofless than about 10⁻⁶ to bind to each other, but not with other captureagents or targets.

The first and second capture agents may be moieties that are capable ofspecifically binding to a target protein or probe of interest,respectively, when both brought into contact with the sample undersuitable reaction conditions. The binding interaction is, in some cases,mediated by an affinity region of the capture agent and a complementaryaffinity region of the target protein or probe. Any convenient captureagents may be selected as first and second capture agents and utilizedto specifically bind a target analyte or a probe into a complex. Aspectsof the subject methods include producing a complex of a probe-labelledglycosylated target protein that is specifically bound to both the firstand second capture agents.

In some cases, the capture agent is itself part of a larger complex thatincludes additional components which do not specifically interact withthe target protein. In some cases, one or more components (e.g., asdescribed herein) of the complex are contacted with the sample andspecifically bind to the first or second capture agent.

Capture agents of interest include, but are not limited to, proteinssuch as antibodies, scaffolded protein ligands or proteins involved inknown biomolecule interactions (e.g., polynucleotide binding proteins,protein-protein interactions, or avidin-biotin interactions),polynucleotides such as aptamers or polynucleotides with complementarysequences, peptides, enzyme substrates, antigens, haptens, smallmolecules, inhibitors, or an analog thereof. In some embodiments, thefirst capture agent and the second capture agent are independentlyselected from a nucleic acid (e.g., an aptamer or complementarypolynucleotide sequence), a polypeptide (e.g., an antibody), and a smallmolecule (e.g., a hapten). In some cases, the first capture agent andthe second capture agent are independently selected from a nucleic acid,a protein, a peptide, or a small molecule (e.g., an antibody, a hapten,an aptamer, etc).

Target-specific capture agents may have a variety of structures providedthat they are capable of specifically binding to a target protein orprobe of interest under suitable reaction conditions. For example, wherethe target molecule is a nucleic acid, a suitable target-specificcapture agent may be a nucleic acid molecule having a region of sequencecomplementarity to a region of the target nucleic acid molecule, e.g., aregion of substantial or absolute sequence complementarity. Where thetarget is a protein or fragment thereof a suitable target-specificcapture agent may be an antibody capable of specifically binding to thetarget molecule. For example, where the target moiety is a probe such asa biotin moiety, a suitable target-specific capture agent may be ananti-biotin antibody or may be an avidin moiety (i.e., member of a pairof specific binding moieites that specifically binds a biotin moiety andis the protein family that includes avidin), such as avidin,streptavidin or neutravidin protein, or a derivative thereof. In certainembodiments, the first capture agent is an anti-target protein antibodyand the second capture agent is an avidin moiety.

Additional binding members capable of specific interactions are known inthe art and accordingly a suitable target-specific capture agent may bereadily identified and prepared for a specific target molecule oranalyte of interest using standard techniques.

Nucleic Acid Tags

Aspects of the present disclosure include specifically binding to aglycosylated target protein first and second conjugates comprising firstand second nucleic acid tags, respectively. As used herein, the term“nucleic acid tag” refers to a polynucleotide that has a particularsequence which may be used to identify the analyte to which it isconnected or bound. The first and second nucleic acid tags are selectedsuch that they are together capable of hybridizing to a particularbridging nucleic acid to produce an amplicon (e.g., as describedherein). In some cases, the set of nucleic acids including the first andsecond nucleic acid tags and the bridging nucleic acid together arecapable or forming a nucleic acid complex that defines a unique ampliconwhich finds use in the subject methods to detect and/or quantitate theglycosylated target protein of interest.

Any convenient sequences of nucleic acids may be selected for use in thesubject first and second nucleic acid tags to provide for detection ofthe target glycosylated target protein. The nucleic acid tags may be ofany convenient length. In some instances, the nucleic acid tag is atleast 6 nucleotides in length, including but not limited to e.g., atleast 10 nucleotides in length, at least 15 nucleotides in length, atleast 16 nucleotides in length, at least 17 nucleotides in length, atleast 18 nucleotides in length, at least 19 nucleotides in length, atleast 20 nucleotides in length, at least 25 nucleotides in length, atleast 30 nucleotides in length, and may be as long as 60 nucleotides inlength or longer, where the length of the nucleic acid tags willgenerally range from 10 to 50 nucleotides in length, including but notlimited to, e.g., from about 15 to 50 nucleotides in length, or fromabout 20 to 35 nucleotides in length.

As used herein, the term “bridging nucleic acid” refers to anypolynucleotide that joins two or more separate polynucleotides or twotermini of a single polynucleotide by simultaneously hybridizing withcomplementary regions on each polynucleotide or complementary regions ofthe polynucleotide termini. In certain instances, a bridgingpolynucleotide joins two target protein-bound nucleic acid tags bysimultaneously hybridizing with a first complementary region of a firstnucleic acid tag and a second complementary region of a second nucleicacid tag. Bridging polynucleotides may be partially or completely singlestranded, including partially single stranded and partially doublestranded.

A bridging nucleic acid may “bridge” two or more polynucleotides to forma polynucleotide complex. As used herein, the terms “polynucleotide”,“oligonucleotide” and “nucleic acid” are used interchangeably. In someinstances, a bridging polynucleotide may hybridize with twopolynucleotide termini, including termini of the same or differentnucleic acids, such that the termini are adjacent within thepolynucleotide complex, e.g., allowing for the ligation of the adjacenttermini. In some instances, a bridging polynucleotide may hybridize withtwo polynucleotide termini, including termini of the same or differentnucleic acids, such that the termini are not adjacent in the resultingpolynucleotide complex, e.g., are not adjacent such that they cannot bedirectly ligated together. In some instances, e.g., where two termini ofa polynucleotide complex are not adjacent, a splint polynucleotide maybe hybridized in the space between the two termini such that the ends ofthe splint polynucleotide are located adjacent to one or more of thetermini. The term “splint polynucleotide” or “splint nucleic acid” asused herein refers to a polynucleotide, which may generally be singlestranded or partially single stranded and partially double stranded,which may be used to fill one or more gaps between two polynucleotidetermini of a polynucleotide complex, e.g., those complexes formed by useof a bridging polynucleotide. In some instances, a splint polynucleotidemay have complementarity to one or more portions of a bridgingpolynucleotide. In some instances, the termini of one or morepolynucleotides adjacent to a splint polynucleotide may be ligated tothe splint polynucleotide.

Amplification

Proximity ligation assays (PLA) leverage the amplification power of thepolymerase chain reaction (PCR) by linking the presence of the targetanalytes to the production of a PCR amplicon (e.g., as defined herein)which can be detected down to several hundred molecules. Any convenientPLAs may be adapted for use in the subject methods.

An exemplary workflow for the subject Glyco-seq method is shown in FIG.3. Cell lysate is treated via the “Click-it” method to install biotin orother moiety of interest onto O-GlcNAc, followed by incubation with twoantibody-DNA conjugates: one that binds the target protein, and one thatbinds biotin. The two binding events bring the DNA strands into closeproximity where addition of a complementary bridging DNA and DNA ligasecan join them together, generating a PCR amplicon that can be quantifiedby PCR. In some cases, where the pair of capture agents (e.g.,antibodies) bind at nearby sites can the amplicon be constituted, thusleading to specific detection of O-GlcNAc on a glycosylated targetprotein of interest.

Upon formation of an amplicon, or a joined polynucleotide from which anamplicon may be formed, or an elongated polynucleotide from which anamplicon may be formed, the amplicon may be amplified to generate anamplification product. Any convenient method of amplification may beutilized in generating the amplification product, as described in moredetail below, and may depend upon the particular polynucleotide complexformed and/or particular requirements of the overall detection assay. Asthe formation of the amplicon is dependent on glycosylated targetprotein-mediated binding of the first and second conjugates, thepresence of the amplification product may be indicative of the presenceof the glycosylated target protein and/or the amount of the glycosylatedtarget protein in the sample.

In some instances, amplification may be performed by polymerase chainreaction (PCR). In representative PCR amplification reactions, thereaction mixture generally includes a template nucleic acid which iscombined with one or more primers that are employed in the primerextension reaction, e.g., the PCR primers (such as forward and reverseprimers employed in geometric (or exponential) amplification or a singleprimer employed in a linear amplification). As such, in some instances,the hybridized portions of the above described nucleic acid complexesmay serve as “primer” for the amplification reaction. For example, ininstances where linear amplification is employed a single free3′-terminus of hybridized nucleic acid of an above described nucleicacid complex may serve as a primer for amplification. In some instances,one or more additional nucleic acids may be added to serve as primer ina formed nucleic acid complex. For example, in some instances two targetprotein-bound nucleic acid tags may be joined in a ligation reaction andtwo additional primers may be added to facilitate amplification of thenewly ligated nucleic acid segment or template. In some instances, asingle free 3′-terminus of hybridized nucleic acid of an above describednucleic acid complex may serve as a first primer and a second primer maybe added to facilitate amplification.

Any oligonucleotide primers with which the template nucleic acid(hereinafter referred to as template DNA for convenience) is contactedwill be of sufficient length to provide for hybridization tocomplementary template DNA under annealing conditions. The primers willgenerally be at least 6 bp in length, including but not limited to e.g.,at least 10 bp in length, at least 15 bp in length, at least 16 bp inlength, at least 17 bp in length, at least 18 bp in length, at least 19bp in length, at least 20 bp in length, at least 21 bp in length, atleast 22 bp in length, at least 23 bp in length, at least 24 bp inlength, at least 25 bp in length, at least 26 bp in length, at least 27bp in length, at least 28 bp in length, at least 29 bp in length, atleast 30 bp in length, and may be as long as 60 bp in length or longer,where the length of the primers will generally range from 18 to 50 bp inlength, including but not limited to, e.g., from about 20 to 35 bp inlength. In some instances, the template DNA may be contacted with asingle primer or a set of two primers (forward and reverse primers),depending on whether primer extension, linear or exponentialamplification of the template DNA is desired. Methods of PCR that may beemployed in the subject methods include but are not limited to thosedescribed in U.S. Pat. Nos. 4,683,202; 4,683,195; 4,800,159; 4,965,188and 5,512,462, the disclosures of which are herein incorporated byreference.

In addition to the above components, a PCR reaction mixture produced inthe subject methods may include a polymerase and deoxyribonucleosidetriphosphates (dNTPs). The desired polymerase activity may be providedby one or more distinct polymerase enzymes. In many embodiments, thereaction mixture includes at least a Family A polymerase, whererepresentative Family A polymerases of interest include, but are notlimited to: Thermus aquaticus polymerases, including the naturallyoccurring polymerase (Taq) and derivatives and homologues thereof, suchas Klentaq (as described in Proc. Natl. Acad. Sci USA (1994)91:2216-2220, the disclosure of which is incorporated herein byreference in its entirety); Thermus thermophilus polymerases, includingthe naturally occurring polymerase (Tth) and derivatives and homologuesthereof, and the like. In certain embodiments where the amplificationreaction that is carried out is a high fidelity reaction, the reactionmixture may further include a polymerase enzyme having 3′-5′ exonucleaseactivity, e.g., as may be provided by a Family B polymerase, whereFamily B polymerases of interest include, but are not limited to:Thermococcus litoralis DNA polymerase (Vent) (e.g., as described inPerler et al., Proc. Natl. Acad. Sci. USA (1992) 89:5577, the disclosureof which is incorporated herein by reference in its entirety);Pyrococcus species GB-D (Deep Vent); Pyrococcus furiosus DNA polymerase(Pfu) (e.g., as described in Lundberg et al., Gene (1991) 108:1-6, thedisclosure of which is incorporated herein by reference in itsentirety), Pyrococcus woesei (Pwo) and the like. Generally, the reactionmixture will include four different types of dNTPs corresponding to thefour naturally occurring bases are present, i.e. dATP, dTTP, dCTP anddGTP and in some instances may include one or more modified nucleotidedNTPs.

A PCR reaction will generally be carried out by cycling the reactionmixture between appropriate temperatures for annealing,elongation/extension, and denaturation for specific times. Suchtemperature and times will vary and will depend on the particularcomponents of the reaction including, e.g., the polymerase and theprimers as well as the expected length of the resulting PCR product. Insome instances, e.g., where nested or two-step PCR are employed thecycling-reaction may be carried out in stages, e.g., cycling accordingto a first stage having a particular cycling program or using particulartemperature(s) and subsequently cycling according to a second stagehaving a particular cycling program or using particular temperature(s).

In some instances, amplification may be carried out under isothermalconditions, e.g., by means of isothermal amplification. Methods ofisothermal amplification generally make use of enzymatic means ofseparating DNA strands to facilitate amplification at constanttemperature, such as, e.g., strand-displacing polymerase or a helicase,thus negating the need for thermocycling to denature DNA. Any convenientand appropriate means of isothermal amplification may be employed in thesubject methods including but are not limited to: loop-mediatedisothermal amplification (LAMP), strand displacement amplification(SDA), helicase-dependent amplification (HDA), nicking enzymeamplification reaction (NEAR), and the like. LAMP generally utilizes aplurality of primers, e.g., 4-6 primers, which may recognize a pluralityof distinct regions, e.g., 6-8 distinct regions, of target DNA.Synthesis is generally initiated by a strand-displacing DNA polymerasewith two of the primers forming loop structures to facilitate subsequentrounds of amplification. LAMP is rapid and sensitive. In addition, themagnesium pyrophosphate produced during the LAMP amplification reactionmay, in some instances be visualized without the use of specializedequipment, e.g., by eye. SDA generally involves the use of astrand-displacing DNA polymerase (e.g., Bst DNA polymerase, Large(Klenow) Fragment polymerase, Klenow Fragment (3′-5′ exo-), and thelike) to initiate at nicks created by a strand-limited restrictionendonuclease or nicking enzyme at a site contained in a primer. In SDAthe nicking site is generally regenerated with each polymerasedisplacement step, resulting in exponential amplification. HDA generallyemploys: a helicase which unwinds double-stranded DNA unwinding toseparate strands; primers, e.g., two primers, that may anneal to theunwound DNA; and a strand-displacing DNA polymerase for extension. NEARgenerally involves a strand-displacing DNA polymerase that initiateselongation at a nicks, e.g., created by a nicking enzyme. NEAR is rapidand sensitive, quickly producing many short nucleic acids from a targetsequence.

In some instances, entire amplification methods may be combined oraspects of various amplification methods may be recombined to generate ahybrid amplification method. For example, in some instances, aspects ofPCR may be used, e.g., to generate the initial template or amplicon orfirst round or rounds of amplification, and an isothermal amplificationmethod may be subsequently employed for further amplification. In someinstances, an isothermal amplification method or aspects of anisothermal amplification method may be employed, followed by PCR forfurther amplification of the product of the isothermal amplificationreaction.

In some instances, the amplification step and the detection step,described below, may be combined. In some instances, the particularamplification method employed allows for the qualitative detection ofamplification product, e.g., by visual inspection of the amplificationreaction with or without a detection reagent. In one embodiment, targetprotein-bound nucleic acid complex is amplified by isothermalamplification, e.g., LAMP, and the amplification generates a visualchange in the amplification reaction indicative of efficientamplification and thus presence of the glycosylated target protein inthe sample. In some instances, the amplification and detection steps arecombined by monitoring the amplification reaction during amplificationsuch as is performed in, e.g., real-time PCR (RT-PCR), also referred toherein as quantitative PCR (qPCR), and described in more detail below.

In some instances, the methods described herein may make use of thosemethods, e.g., amplification methods, and components thereof, employedin proximity ligation assays (PLA) and proximity elongation assays (PEA)including but not limited to, e.g., rolling circle amplification (RCA),binding-induced DNA assembly (BINDA), nicking enzyme assistedfluorescence signal amplification (NEFSA), and, e.g., those described inJanssen et al. (2013) Sensors, 13, 1353-1384, the disclosure of which isincorporated herein by reference in its entirety.

Detection

A variety of technologies are available to detect nucleic acid productswhich may be adapted for use in the subject methods. In some cases,fluorescence-based quantitative PCR (qPCR) may be used forhigh-throughput detection of DNA and RNA. The present disclosureprovides an analytical platform that harnesses the power and ease ofqPCR to detect glycosylation on a protein of interest. This method,which is termed herein Glyco-seq, makes use of “Click-it” labeling andproximity ligation assay and provides for detection of O-GlcNAc onproteins of interest by PCR. The subject methods can provide for thehighly sensitive, multiplexed detection of O-GlcNAcylated proteins fromcell lysate without enrichment while using accessible, affordable, andfamiliar qPCR equipment and reagents.

The presence of the amplification product may be determined, includingqualitatively determined or quantitatively determined, by any convenientmethod. In some instances, the presence of the amplification product maybe qualitatively determined, e.g., through a physical change in theamplification reaction that is indicative of efficient amplification ofthe target polynucleotide complex.

In some instances, the amplification product is detected and/or theamount of amplification product is measured by a detection protocol fornon-specific detection of the amplified nucleic acid or a protocol forspecific detection of the amplified nucleic acid. Representativenon-specific detection protocols of interest include protocols thatemploy signal producing systems that selectively detect double strandednucleic acid products, e.g., via intercalation. Representativedetectable molecules that find use in such embodiments includefluorescent nucleic acid stains, such as phenanthridinium dyes,including monomers or homo- or heterodimers thereof, that provideenhanced fluorescence when complexed with nucleic acids. Examples ofphenanthridinium dyes include ethidium homodimer, ethidium bromide,propidium iodide, and other alkyl-substituted phenanthridinium dyes. Inanother embodiment, a nucleic acid stain includes an acridine dye, or ahomo- or heterodimer thereof, such as acridine orange, acridinehomodimer, ethidium-acridine heterodimer, or9-amino-6-chloro-2-methoxyacridine. In yet another embodiment, thenucleic acid stain is an indole or imidazole dye, such as Hoechst 33258,Hoechst 33342, Hoechst 34580, DAPI (4′,6-diamidino-2-phenylindole) orDIPI (4′,6-(diimidazolin-2-yl)-2-phenylindole). Other permitted nucleicacid stains include, but are not limited to, 7-aminoactinomycin D,hydroxystilbamidine, LDS 751, selected psoralens (furocoumarins), styryldyes, metal complexes such as ruthenium complexes, and transition metalcomplexes (incorporating Tb³⁺ and Eu³⁺, for example). In certainembodiments of the invention, the nucleic acid stain is a cyanine dye ora homo- or heterodimer of a cyanine dye that gives an enhancedfluorescence when associated with nucleic acids. In some instances, dyesdescribed in U.S. Pat. No. 4,883,867, U.S. Pat. No. 5,582,977, U.S. Pat.No. 5,321,130, and U.S. Pat. No. 5,410,030, which are incorporatedherein by reference in their entirety, may be used, including nucleicacid stains commercially available under the trademarks TOTO, BOBO,POPO, YOYO, TO-PRO, BO-PRO, PO-PRO and YO-PRO (Life Technologies, Inc.Grand Island, N.Y.). In some instances, dyes described in U.S. Pat. No.5,436,134, U.S. Pat. No. 5,658,751 and U.S. Pat. No. 5,863,753, whichare incorporated herein by reference in their entirety, may be used,including nucleic acid stains commercially available under thetrademarks SYBR, SYTO, SYTOX, PICOGREEN, OLIGREEN, and RIBOGREEN (LifeTechnologies, Inc. Grand Island, N.Y.). In yet other embodiments of theinvention, the nucleic acid stain is a monomeric, homodimeric orheterodimeric cyanine dye that incorporates an aza- orpolyazabenzazolium heterocycle, such as an azabenzoxazole,azabenzimidazole, or azabenzothiazole, that gives enhanced fluorescencewhen associated with nucleic acids, including nucleic acid stainscommercially available under the trademarks SYTO, SYTOX, JOJO, JO-PRO,LOLO, LO-PRO (Life Technologies, Inc. Grand Island, N.Y.).

In yet other embodiments, a signal producing system that is specific forthe amplification product, as opposed to double stranded molecules ingeneral, may be employed to detect the amplification. In theseembodiments, the signal producing system may include a probe nucleicacid that specifically binds to a sequence found in the amplificationproduct, where the probe nucleic acid may be labeled with a directly orindirectly detectable label. A directly detectable label is one that canbe directly detected without the use of additional reagents, while anindirectly detectable label is one that is detectable by employing oneor more additional reagent, e.g., where the label is a member of asignal producing system made up of two or more components. In someembodiments, the label is a directly detectable label, where directlydetectable labels of interest include, but are not limited to:fluorescent labels, radioisotopic labels, chemiluminescent labels, andthe like. In some embodiments, the label is a fluorescent label, wherethe labeling reagent employed in such embodiments is a fluorescentlytagged nucleotide(s), e.g. fluorescently tagged CTP (such as Cy3-CTP,Cy5-CTP) etc. Fluorescent moieties which may be used to tag nucleotidesfor producing labeled probe nucleic acids include, but are not limitedto: fluorescein, the cyanine dyes, such as Cy3, Cy5, Alexa 555, Bodipy630/650, and the like. Other labels, such as those described above, mayalso be employed.

In those embodiments where the signal producing system is a fluorescentsignal producing system, signal detection in some cases includesdetecting a change in a fluorescent signal from the reaction mixture toobtain an assay result. In other words, any modulation in thefluorescent signal generated by the reaction mixture is assessed. Thechange may be an increase or decrease in fluorescence, depending on thenature of the label employed, and in certain embodiments is an increasein fluorescence. The sample may be screened for an increase influorescence using any convenient means, e.g., a suitable fluorimeter,such as a thermostable-cuvette or plate-reader fluorimeter. Fluorescenceis suitably monitored using a known fluorimeter. The signals from thesedevices, for instance in the form of photo-multiplier voltages, are sentto a data processor board and converted into a spectrum associated witheach sample tube. Multiple reaction vessels, e.g., multiple tubes,multi-well plates, etc., can be assessed at the same time.

In some instances, the elongation and/or amplification of a particularpolynucleotide of a nucleic acid complex, e.g., a target protein-boundnucleic acid complex, a bridging polynucleotide, a circularizingoligonucleotide, etc., results in the duplication of one or morespecific nucleic acid sequences resulting in one or more strandscontaining repeats of the one or more specific nucleic acid sequences.Such repetitive sequences may be detected, e.g., through hybridizationof a probe nucleic acid specific for the repeated specific sequence. Incertain instances, a tagged probe nucleic acid, e.g., a fluorescentlytagged probe nucleic acid, an enzymatically tagged probe nucleic acid, aradiolabel tagged probe nucleic acid, etc., specific for the repeatedspecific sequence may be utilized to detect an elongated polynucleotideor amplification product that contains the repeated specific sequence.In some instances, hybridization of a tagged probe nucleic acid to arepeating sequence of an elongated polynucleotide or amplificationproduct allows for the detection of the elongated polynucleotide oramplification product due to the high number of tagged probe nucleicacids hybridized to the elongated polynucleotide or amplificationproduct, which results in a high local concentration of detectable tag.

For example, in some instances, repeats of one or more sequences of atarget protein-bound nucleic acid complex are contained in anamplification product or elongation product produced according to themethods described herein and the repeats are detected through the use ofa tagged probe nucleic acid specific for the repeating sequence units.In some instances, repeats of one or more sequences of a bridgingpolynucleotide are contained in an amplification product or elongationproduct produced according to the methods described herein and therepeats are detected through the use of a tagged probe nucleic acidspecific for the repeating sequence units. In some instances, repeats ofone or more sequences of a circularizing oligonucleotide are containedin an amplification product or elongation product produced according tothe methods described herein and the repeats are detected through theuse of a tagged probe nucleic acid specific for the repeating sequenceunits.

In certain embodiments, a repeating nucleic acid sequence may beproduced by one or more of the elongation and/or amplification methodsdescribed herein, e.g., PCR amplification, isothermal amplification(e.g., RCA), etc., and the elongation and/or amplification product maybe made detectable through hybridization of one or more fluorescentlylabeled probe nucleic acid to the elongation and/or amplificationproduct. Such detectable elongation and/or amplification product may beidentified through any convenient means for detecting fluorescence,including but not limited to, e.g., fluorescent microscopy, flowcytometry, imaging flow cytometry, etc. In some instances,identification of a detectable elongation and/or amplification productmay allow for detection or identification of a molecule, particle, cell,tissue, organism, etc., associated with the antigen binding agent of thecomplex from which the elongation and/or amplification product wasderived. For example, in some instances, fluorescent probe-boundelongation and/or amplification product may remain associated with acell that produced the antigen binding agent allowing identification ofthe cell, e.g., by fluorescent microscopy, and/or isolation of the cell,e.g., by fluorescent activated cell sorting (FACS).

As noted above, in some instances, amplification may be monitored inreal time to provide detection and/or quantitation. Where the detectionprotocol is a real time protocol, e.g., as employed in RT-PCR or qPCRreaction protocols, data may be collected at frequent intervals, forexample once every 10 milliseconds (ms), or more or less frequently thanonce every 10 ms, throughout the reaction. By monitoring thefluorescence of the reactive molecule from the sample during each cycle,the progress of the amplification reaction can be monitored in variousways. For example, the data provided by melting peaks can be analyzed,for example by calculating the area under the melting peaks and thesedata plotted against the number of cycles.

The spectra generated in this way can be resolved, for example, using“fits” of pre-selected fluorescent moieties such as dyes, to form peaksrepresentative of each signaling moiety (i.e. fluorophore). The areasunder the peaks can be determined which represents the intensity valuefor each signal, and if required, expressed as quotients of each other.The differential of signal intensities and/or ratios will allow changesin labeled probes to be recorded through the reaction or at differentreaction conditions, such as temperatures. The changes are related tothe binding phenomenon between the oligonucleotide probe and the targetsequence or degradation of the oligonucleotide probe bound to the targetsequence. The integral of the area under the differential peaks willallow intensity values for the label effects to be calculated.

Screening the mixture for a change in fluorescence provides one or moreassay results, depending on whether the sample is screened once at theend of the amplification reaction, or multiple times during thereaction, e.g., after each cycle (e.g., as is done in RT-PCRmonitoring).

According to the methods described herein, the presence of glycosylatedtarget protein may be detected, e.g., as above or below a particulardetection threshold, or may be measured, e.g., the actual amount orconcentration of the glycosylated target protein in the sample may bemeasured when present above a particular detection threshold. The actualdetection threshold for a subject glycosylated target protein detectionreaction will vary and will depend on, e.g., the glycosylated targetprotein to be detected the particular amplification method employed, thedetection method employed, and the like. In some instances, thedetection threshold for the subject detection methods may range from 15ng/ml to 1 pg/ml and may include less than 15 ng/ml, less than 14 ng/ml,less than 13 ng/ml, less than 12 ng/ml, less than 11 ng/ml, less than 10ng/ml, less than 9 ng/ml, less than 8 ng/ml, less than 7 ng/ml, lessthan 6 ng/ml, less than 5 ng/ml, less than 4 ng/ml, less than 3 ng/ml,less than 2 ng/ml, less than 1 ng/ml, less than 500 pg/ml, less than 400pg/ml, less than 300 pg/ml, less than 200 pg/ml, less than 100 pg/ml,less than 90 pg/ml, less than 80 pg/ml, less than 70 pg/ml, less than 60pg/ml, less than 50 pg/ml, less than 40 pg/ml, less than 35 pg/ml, lessthan 30 pg/ml, less than 25 pg/ml, less than 20 pg/ml, less than 19pg/ml, less than 18 pg/ml, less than 17 pg/ml, less than 16 pg/ml, lessthan 15 pg/ml, less than 14 pg/ml, less than 13 pg/ml, less than 12pg/ml, less than 10 pg/ml, etc. In some instances, the detectionthreshold for a particular detection method described herein may beexpressed in the minimum moles of glycosylated target protein that maybe detected in a sample and, such detection thresholds may range from200 attomoles to 100 zeptomoles, including but not limited to e.g., 200attomoles, 190 attomoles, 180 attomoles, 170 attomoles, 160 attomoles,150 attomoles, 140 attomoles, 130 attomoles, 120 attomoles, 110attomoles, 100 attomoles, 90 attomoles, 80 attomoles, 70 attomoles, 60attomoles, 50 attomoles, 40 attomoles, 30 attomoles, 20 attomoles, 10attomoles, 1 attomole, 900 zeptomoles, 800 zeptomoles, 700 zeptomoles,600 zeptomoles, 500 zeptomoles, 400 zeptomoles, 350 zeptomoles, 300zeptomoles, 250 zeptomoles, 200 zeptomoles, 190 zeptomoles, 180zeptomoles, 170 zeptomoles, 160 zeptomoles, 150 zeptomoles, 140zeptomoles, 130 zeptomoles, 120 zeptomoles, 110 zeptomoles, 100zeptomoles, etc.

Following detection, which may or may not include qualitative orquantitative measurement of the amplification product, the result of thedetection may be assessed to determine the likelihood that theglycosylated target protein is present in the sample. In making suchassessments, in some instances, the subject reaction may be compared toone or more control reactions or reference values. Control reactions ofthe subject method include positive controls, e.g., a sample known tocontain the target protein of interest and/or known to contain a knownamount of target protein of interest and/or known to have a particularlevel of glycosylation. Control reactions may also include negativecontrols, e.g., samples known to not contain a critical component, e.g.,the target protein, glycosylated target protein, the polymerase, acritical polynucleotide, etc. Reference values to which results of adetection reaction may be compared include but are not limited to areference measurement from any control reaction performed previously, astandard curve gathered from a control reaction, a set of measuredfluorescent values from positive or negative controls, user-definedreference values, manufacturer supplied reference values, etc. In someinstances, assessment of a subject reaction may include comparison to ascale, e.g., a scale of reference values, which can be used to estimatethe amount of antigen binding agent present in the sample.

The subject methods may be used in glycoproteomics to deconvolute therelative signal contributions from changes in target protein abundanceversus changes in the amount of O-GlcNAc present on the protein. Inother methods, an increase in signal detected from O-GlcNAc couldreflect for instance an increase in the modification present perprotein, or a simple increase in the protein abundance with no change inthe modification stoichiometry. The subject Glyco-seq methods are wellequipped to monitor these two parameters independently. Changes inprotein abundance may be monitored side-by-side with changes in O-GlcNAcstoichiometry, and therefore the O-GlcNAc present on proteins ofinterest can be quantified.

Multiplexing

According to the methods described herein, a sample is readily screenedfor the presence of glycosylated target protein. The methods aresuitable for detection of a single glycosylated target protein as wellas multiplex analyses, in which two or more different glycosylatedtarget protein are assayed in the sample. In these latter multiplexsituations, the number of different sets of first and second conjugatesand bridging nucleic acids that may be employed typically ranges fromabout 2 to about 20 or higher, e.g., as up to 100 or higher, 1000 orhigher, etc. In one embodiment, a multiplexed assay may make use ofvarious different capture agents conjugated to unique nucleic acid tags(i.e., conjugates, as described herein) in conjunction with bridgingnucleic acids such that amplification of a particularly unique ampliconis indicative of the presence of the associated glycosylated targetprotein. Accordingly, the subject assays may make use of nucleic acidtagging and/or “barcoding” strategies to allow for the detection and/orquantification of a plurality of glycosylated target proteins in asample. The number of different first and second conjugates, uniquelytagged with nucleic acid barcodes, that may be included in a multiplexedassay as described herein may vary and may be limited only by, e.g., theavailable length of polynucleotide in the first and second conjugatesfor the barcode, the physical limit of conjugate concentration that maybe present in the reaction without negatively impacting the specificbinding to the target protein and/or polynucleotide binding, and thelike.

As such, in some instances, a panel of glycosylated target proteins maybe screened in a single reaction and the presence, quantities or levelof glycosylation of each glycosylated target protein on the panel may beassessed. The detection methods described above may be utilized inparallel for the detection and measurement of amplification products ina duplexed assay. In some instances, in both multiplexed andnon-multiplexed assays, nucleic acid sequencing methods may be utilizedfor detection and/or measurement of amplification product. For example,in some instances, quantitative sequencing may be utilized, e.g., in amultiplexed assay having produced a plurality of amplification products,to determine the relative amounts or presence of each amplificationproduct allowing for a highly sensitive and highly multiplexedassessment of many different glycosylated target proteins in a singlesample.

Aspects of the present disclosure also include methods for detecting alow-abundance protein in a biological sample. The phrase “low-abundanceprotein”, as used herein, refers to one or more proteins (e.g.,glycosylated proteins) present in a sample in a sufficiently lowquantity that they may be difficult to detect by some methods (e.g.,LC-MS/MS approaches that select only the most intense ions in a givensample for fragmentation and/or further analysis). Low abundanceproteins (e.g., low abundance glycosylated proteins) have aconcentration that is less than that of high abundance proteins. Forexample, low abundance proteins may have a concentration of less than100 ng/mL, such as less than 75 ng/mL, such as less than 50 ng/mL andincluding less than 25 ng/mL in a biological sample. In otherembodiments low abundance proteins are present in a biological samplecontaining a mixture of proteins in an amount that is less than or equalto 1000 pg/mg of total protein in the biological sample, such as 750pg/mg, such as 500 pg/mg and including equal to or less than 250 pg/mgof total protein in the biological sample. In certain instances, methodsof the present disclosure include detecting and identifying lowabundance proteins in a biological sample present in an amount that isequal to or less than 100 pg/mg of total protein, less than 50 pg/mg oftotal protein, or less than 10 pg/mg total protein.

Determining the Level of Total Target Protein

In any of the above-described embodiments, a method of the presentdisclosure can further include a step of detecting total target protein(total target protein, including unglycosylated target protein andglycosylated target protein).

For example, in some cases, a capture agent that binds the targetprotein (e.g., that binds an epitope comprising a stretch of aminoacids, such as from 2 amino acids to 20 amino acids) can be used todetermine the amount of total target protein. In some cases, the samecapture agent that is used in the first conjugate, described above, canbe used to determine the level of total target protein. In some cases,the capture agent that is used in the first conjugate, described above,is different from the capture agent(s) used to determine the level oftotal target protein.

Proximity Ligation Assays

In some cases, determining the amount of total target protein is carriedout using a proximity-based ligation assay comprising: (a) contactingthe sample with: (i) a third conjugate comprising a third nucleic acidtag linked to a third capture agent that specifically binds a firstepitope in the target protein; (ii) a fourth conjugate comprising afourth nucleic acid tag linked to a fourth capture agent thatspecifically a second epitope in the target protein; and (iii) abridging nucleic acid that hybridizes to the third and fourth nucleicacid tags; under conditions sufficient to specifically bind the thirdand fourth capture agents to the probe-labeled target protein and tohybridize the bridging nucleic acid to the third and fourth nucleic acidtags to produce a target protein-bound nucleic acid complex; and (b)detecting the target protein-bound nucleic acid complex.

In some cases, the third capture agent and the fourth capture agents areantibodies that recognize distinct epitopes on the target protein.Suitable nucleic acid tags are as described above. Suitable PCR-baseddetection methods are as described above.

Where detection of the total target protein and detection ofglycosylated target protein are both carried out using a proximityligation assay, detection of total target protein and detection ofglycosylated target protein can be carried out in the same reactionvessel. Where detection of the total target protein and detection ofglycosylated target protein are both carried out using a proximityligation assay, detection of total target protein and detection ofglycosylated target protein can be carried out in two separate reactionvessels.

In some cases, the amount of total target protein is compared to theamount of glycosylated target protein, such that a ratio of glycosylatedtarget protein to total target protein is obtained. In some cases, theamount of glycosylated target protein and the amount of total targetprotein are determined over time, e.g., in response to a stimulus.

Immunological Assays

For example, in some cases, the first capture agent is an antibody thatspecifically binds a protein epitope in the target protein. The totalamount of target protein can be determined using a detectably labeledantibody that specifically binds a protein epitope in the targetprotein. In some cases, the antibody is the same as the first captureagent. The amount of total target protein can be determined using, e.g.,an immunological assay, where suitable immunological assays include,e.g., an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay(RIA), and the like.

In some cases, the step of detecting or determining the total targetprotein comprises contacting the sample with a detectably labeledantibody that specifically binds a protein epitope in the targetprotein; forming a complex between the detectably labeled antibody andthe target protein in the sample; and determining the amount of totaltarget protein based on the amount of detectably labeled antibody in thecomplex. Suitable detectable labels include, e.g., radioisotopes;enzymes that generate fluorescent products, luminescent products, orcolored products; fluorescent proteins; fluorescent dyes; and the like.

In some cases, the amount of total target protein is compared to theamount of glycosylated target protein, such that a ratio of glycosylatedtarget protein to total target protein is obtained. In some cases, theamount of glycosylated target protein and the amount of total targetprotein are determined over time, e.g., in response to a stimulus.

Utility

The methods and compositions described herein have particular utility inthe detection and/or quantification of a glycosylated target proteinpresent in a sample. Such detection may find various applications in avariety of technological fields including but not limited to e.g., basicscientific research (e.g., biomedical research, biochemistry research,immunological research, molecular biology research, microbiologicalresearch, cellular biology research, genetics, and the like), medicaland/or pharmaceutical research (e.g., drug discovery research, drugdesign research, drug development research, pharmacology, toxicology,medicinal chemistry, pre-clinical research, clinical research,personalized medicine, and the like), medicine, epidemiology, publichealth, biotechnology, veterinary science, veterinary medicine,agriculture, material science, molecular detection, moleculardiagnostics, and the like.

In some instances, methods described herein find use in detection ofglycosylated target protein in a biological sample from a subject. Theterm “subject” as used herein refers to an animal, including humans,livestock, pets, laboratory animals, bioproduction animals (e.g.,animals used to generate a bioproduct, e.g., an antibody), and the like.In some instances, a sample is derived from a mammalian subject,including e.g., mammalian tissue, mammalian cells, mammalian bodilyfluid, mammalian excreted bodily fluids, mammalian semi-solidsecretions, and the like.

Compositions

Aspects of the present disclosure include compositions, e.g., reagentsand kits, useful in practicing the methods described herein. Any of thecomposition components described herein may find use individually in amethod or kit for detecting glycosylate target proteins. For example,the present disclosure provides first and second conjugates useful inthe described detection methods.

In some embodiments, the composition includes (a) a first conjugateincluding a first nucleic acid tag linked to a first capture agent(e.g., as described herein) that is capable of specifically binding atarget protein; and (b) a second conjugate including a second nucleicacid tag linked to a second capture agent (e.g., as described herein)that is capable of specifically binding a probe. The compositions mayfurther include a bridging nucleic acid that is complementary to thefirst and second nucleic acid tags (e.g., as described herein). Thecomposition may further includes a probe-labeled glycosylated targetprotein (e.g., as described herein).

Kits

In yet another aspect, the present disclosure provides kits forpracticing the subject methods, e.g., as described above. The subjectkits may include any combination of the herein described reagents, orcompositions useful in practicing the methods as described aboveincluding but not limited to, e.g., one or more of the described firstand second conjugates, bridging polynucleotides, splint polynucleotides,reactive probes, enzymatic reagents (e.g., ligases), and the like.Subject kits may further include one or more reagent preparationreagents including but not limited to, e.g., reagents for labelling anmetabolically tagged target protein, reagents for functionalizing apolynucleotide, reagents for conjugation of a polynucleotide and/or acapture agent. In addition, subject kits may further include assayreagents or reagents useful in performing an assay of a sample, e.g., apatient sample, to allow for an assessment, e.g., of whether one or moreglycosylated target proteins are present in a sample from the subject.Such assay reagents may include but are not limited to, e.g., detectionreagents, sample preparation reagents, amplification reagents (e.g., PCRreagents and/or isothermal amplification reagents and/or qPCR reagents,etc.) and binding reagents (e.g., conjugates, and the like), buffers,diluents, etc. Such assay kits may further include sample collectioncomponents, e.g., sample collection containers and/or sample collectiondevices, etc. The above components may be present in separate containersor one or more components may be combined into a single container, e.g.,a glass or plastic vial or tube.

Kits may further include control reagents and samples including but notlimited to, e.g., control samples (e.g., positive control samples,negative control samples, etc.) calibration reagents (e.g., fluorescentcalibration reagents, etc.).

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, removable drive (e.g., flash memory device), etc.,on which the information has been recorded. Yet another means that maybe present is a website address which may be used via the internet toaccess the information at a removed site. Any convenient means may bepresent in the kits.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1: Glyco-seq to Detect O-GlcNAcylation State of a Target ProteinDesign and Synthesis of Reagents for Detection of O-GlcNAc by ProximityLigation

There are many published protocols that are suitable for proximityligation. In preliminary experiments, succinimidyl4-[N-maleimidomethyl]-cyclohexane-1 carboxylate (SMCC) crosslinkingreagent was used for generating antibody-DNA conjugates (FIG. 4). SMCCis a lysine-to-sulfhydryl crosslinker. The succinimidyl ester reactswith lysine residues on the antibody to attach maleimide groups forlater functionalization with thiolated oligonucleotides. The thiolatedoligonucleotides used in proximity ligation assay (PLA) areapproximately 50 base pairs in length. The SMCC crosslinker is ascaleable reagent used in the preparation of antibody conjugations. Thecovalent conjugation of DNA to several antibodies using SMCC wasconfirmed by a mass shift on sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) gel. Excess DNA was used, and the conjugatewas purified to remove the excess DNA. The DNA:antibody ratio was onaverage 2.5 oligos per antibody as determined by UV-VIS. These syntheseswere performed on a 20 μg scale with a ˜50% yield, providing enoughreagent for ca. 30,000 assays. Several conjugation strategies werescreened to achieve a combination of high yield, ease, affordability,and reproducibility for these reagents.

Use of Gyco-Seq to Detect O-GlcNAc on Recombinant and EndogenousProteins

The antibody-DNA conjugates are used to detect O-GlcNAc on purifiedproteins using a Glyco-seq method. Alpha crystallin (Ac), a bovineprotein with low levels of O-GlcNAc modification is analyzed. Theprotein is subjected to “Click-it” conditions to append biotin ontoO-GlcNAc, and then treated with two antibody-DNA conjugates: onedirected against biotin the other directed against Ac itself. Upontreatment with a bridging DNA (which is ca. 20 base pairs in length) andligase, and PCR amplification and detection of DNA using a SYBRgreen-based qPCR kit, a signal resulting from amplified DNA is observed.The above described experiments were performed on purified Ac. Purifiedprotein was also added to lysate to test the detection capability of themethod in a more complex environment as shown in FIG. 5A-5B.

Several types of control experiments are also performed. The purifiedprotein is treated with OGA to cleave off the O-GlcNAc, or withheat-deactivated OGA. Signal only results from the sample that stillretains O-GlcNAc. Also, a parallel experiment is performed in which Acis treated with a second antibody-DNA conjugate targeted towards the Acprotein, instead of the anti-biotin conjugate. This latter set ofconjugates is designed to detect the protein itself, and produces signalirrespective of treatment with OGA.

FIG. 5A-5B: Detection of O-GlcNAc in a complex environment.Alpha-crystallin (Ac) was treated with either OGA or heat-killed OGA,and then added into cell lysate at 1% wt, and detected either O-GlcNAc(A) or total protein level (B) by Glyco-seq. FIG. 5A. OGA treated sampleshows a significantly weak signal due to the loss of O-GlcNAc. In FIG.5B, both samples showed strong signal when total protein level wasdetected. This result verifies that the observed signal difference in(A) was due to differential O-GlcNAc levels. (ΔCT: change in cyclethreshold; a standard means of reporting qPCR signal relative to acontrol sample). The results illustrated in FIG. 5A-5B demonstrate thatGlyco-seq may be used for the protein-specific detection of O-GlcNAc.The purified Ac was added to lysate to evaluate the detection capabilityin a more complex environment as shown in FIG. 5A-5B. In was observedthat very little sample was required to detect signal over background.In order to benchmark the sensitivity of this technique versus otheranalytical strategies, a proximity ligation assay was performed inparallel with an anti-biotin Western blot assay directed against the Acsample which had been treated via “Click-it” (FIG. 6). FIG. 6 shows acomparison of Glyco-seq versus Western blot. Glyco-seq signal isreported as ΔCT as described in FIG. 5A-5B. Western blotting wasperformed with streptavidin-HRP. In this experiment, O-GlcNAcylation wasdetected via Glyco-seq down to 0.1 fmol of protein. This is 104-foldmore sensitive than what was observed via Western blotting, whichrequired 1 pmol of sample. This sensitivity is highly advantageous forreduced sample consumption and ease of detection for low-abundancetargets.

Glyco-seq is applied to several other protein targets to demonstrategeneralizability. The excellent sensitivity of Glyco-seq is harnessed todetect the O-GlcNAcylation state of transcription factors. O-GlcNAc isknown to modify a number of transcription factors, and is thought toplay a role in regulating their action. To evaluate the use ofGlyco-seq, the protein c-Rel is analyzed according to the workflowdescribed above. c-Rel is a low copy number transcription factor thatalters expression of cytokine-encoding genes upon O-GlcNAcylation. TheGlyco-seq reagents are used to detect endogenous c-Rel in Jurkatlysates. As a control, selective immunodepletion of c-Rel from thelysate is also be performed. The signal resulting from Glyco-seqdiminishes as c-Rel is depleted.

To verify that the subject assay is accurately reporting changes in theamount of O-GlcNAcylation present on a target protein, several controlexperiments are performed. Cells are treated with Thiamet G, a known OGAinhibitor to check for an increase in O-GlcNAcylation on specificproteins. Treatment with Thiamet G prevents O-GlcNAc from beinghydrolyzed from proteins, leading to an increase in detectableO-GlcNAcylation. The fold-changes observed from measurements using thesubject methods to those found from other methods are compared.SILAC-based mass spectrometry has been used to report the changes ofO-GlcNAcylation on about 30 protein targets, where the stoichiometrychanges can be anywhere between 0.8-25 fold (Zachara et al., The dynamicstress-induced “O-GlcNAc-ome” highlights functions for O-GlcNAc inregulating DNA damage/repair and other cellular pathways. Amino Acids2011, 40(3), 793-808). O-GlcNAcylation is quantified on target proteinsfrom high to low fold changes including SEC24-C (23.8 fold), NF45 (4.3fold), NUP153 (2.1 fold), NUP54 (1.5 fold) and OGT (1.0 fold). Thesestudies establish the minimum fold-change that Glyco-seq can detect.

Example 2: Multiplexed Glyco-Seq as a Platform for the Detection ofProtein O-GlcNAcylation

The subject methods can be used to detect multiple target proteins atonce (e.g., multiplexing). This is accomplished by using several pairsof first and second antibody-DNA conjugates, each directed at differenttarget proteins. Each pair is prepared with unique DNA sequencesincorporated into the first and second conjugates. Primer pairs that aredesigned to uniquely amplify each ligated pair of conjugates are used toachieve detection, e.g., via qPCR. In this manner, multiple targets areanalyzed in a single experiment by simply performing the assay with anew set of primers. Multiplexing is performed in the context ofGlyco-seq for the detection of O-GlcNAc.

Measuring Changes in Protein Abundance Versus Changes in O-GlcNAc

The subject methods find use in glycoproteomic investigations todifferentiate an increase in detected glycosylation arising from anincrease in the amount of glycosylation present on a protein, from anincrease in the amount of the glycoprotein present in a sample. Changesin protein abundance can be detected using paired antibody-DNAconjugates that are both directed at the protein (e.g., to differentprotein epitopes). The amount of amplicon reconstituted by this ligationcorrelates with protein abundance. With appropriate standards andstatistical power, by comparing the signal from the subject Glyco-seqexperiment with the signal from such a PLA for protein detection, theO-GlcNAc present on proteins of interest is quantified. The subjectmethod detects protein abundance in parallel with the amount of O-GlcNAcon the protein.

Assay to Detect Other Modifications and Quantify Changes in Modification

Sites of O-GlcNAc modification are often competitive withphosphorylation in what is often described as the “Yin-Yang” model(Groves et al., Dynamic O-GlcNAcylation and its roles in the cellularstress response and homeostasis. Cell Stress Chaperones 2013, 18(5),535-55). The subject methods provide for the parallel detection ofprotein phosphorylation by incorporating an anti-phosphoserine oranti-phosphotyrosine antibody-DNA conjugate into the Glyco-seq workflowinstead of the anti-biotin/DNA conjugate.

Antibody-DNA conjugates are prepared to provide for simultaneousdetection of O-GlcNAc and phosphorylation on RNA polymerase-II, whichserves many roles in cells including initiation of transcription andrecruitment of the RNA processing machinery, and is reciprocallymodified by these two Post-Translatonal Modifications (PTMs). These PTMsare monitored in lysates derived from cells that have and have not beensubjected to stimuli that alter transcription. Comparing the data fromboth lysate sets reveals changes in O-GlcNAc levels, as well as changesin phosphorylation levels in response to the stimuli. In this manner, inone experiment it is observed how the amount of each modification on RNApolymerase-II changes in response to the same stimuli.

Multiplexed Assay with Model System of In Vitro Glycosylated RecombinantProteins

The subject assays are sensitive for the detection of low abundanceproteins without enrichment. In addition, the subject assay includes theability to detect changes in many proteins at once. Unique primer pairsare used to encode the identity of each protein in a multiplexed assay.

All antibody-DNA conjugates are present in the assay and ligatedsimultaneously using a single universal bridging DNA. Deconvolution isachieved by interrogating with different primer sets via qPCR. Thestrength of this strategy is underscored by the availability ofinexpensive premade 96- and 384-well plates with dried primers. Theligated library pool is then partitioned into the many wells tointerrogate up to 50 targets in a single experiment while only consuminga few ng of cell lysate.

To demonstrate Glyco-seq for the multiplexed detection of O-GlcNAc onseveral proteins, several purified model O-GlcNAcylated proteins areused as a model system for the subject multiplexed method. Rabbitreticulocyte lysate retains endogenous OGT activity and is commonly usedas a method for the in vitro O-GlcNAcylation of proteins (Starr, C. M.;Hanover, J. A. Glycosylation of nuclear pore protein p62. Reticulocytelysate catalyzes O-linked N-acetylglucosamine addition in vitro. J BiolChem 1990, 265(12), 6868-73). The recombinant, unglycosylated proteinsare mixed together in known concentrations and glycosylated with thereticulocyte lysate. The model set is analyzed using the subjectmultiplexed proximity ligation assay described herein, using proximityprobes to detect both glycosylation and protein abundance. Using thismaster mix as a standard, the detection limit and reproducibility of themultiplexed proximity ligation assay is determined.

While the sequence diversity of oligonucleotides is immense, the scopeof multiplexability is in practice limited by the orthogonality of theprimer pairs and cross-reactivity of the DNA on antibody-DNA conjugates,which is determined empirically. To evaluate the primers, theunconjugated amplicons are mixed together in known quantities to createa standard. Next, qPCR is performed using each set of primers and theamplification efficiency and signal intensity compared to ensure thateach set amplifies selectively and in a reproducible manner.

Multiplexed Glyco-Seq Assay to Detect Glycosylation of TranscriptionFactors

In this experiment, antibody-DNA conjugates are synthesized to monitorthe O-GlcNAcylation of several transcription factors.

TABLE 1 selected transcription factors of interest that areO-GlcNAcylated. PDX-1 PGC-1alpha Neuro-D1 ER-alpha C-Rel ER-beta Sp1TORC2/CRTC2 NFkB NFATalpha1 P53 Elf-1 Fox01 c-myc Oct4 Pdx-1 Sox2 C/EBPbeta Stat5a MafA CREB Id2 YY1 USF

Select transcription factors from Table 1 are expressed and purified andglycosylated with reticulocyte lysate as described above to createexternal standards. Next, lysates are prepared by subjecting them to“Click-it” conditions to attach biotin on O-GlcNAc. One set of lysatesis from cells stressed via hypoxia while another set is from untreatedcells as a control. Using the standards, a multiplexed proximityligation experiment is performed to compare the changes inO-GlcNAcylation to reported changes found in transcription factors (seee.g., Ferrer et al., O-GlcNAcylation regulates cancer metabolism andsurvival stress signaling via regulation of the HIF-1 pathway. Mol Cell2014, 54(5), 820-31; and Lazarus et al., HCF-1 is cleaved in the activesite of O-GlcNAc transferase. Science 2013, 342(6163), 1235-9).

This workflow is depicted in FIG. 7. (A) Mix “Click-it” labeled samplewith proximity probes. (B) Ligation of DNA segments that are in closeproximity via a universal connector and ligase. (C) Amplification oftarget specific amplicons by addition samples from (B) into 96-wellprimer plates. (D) Quantification of the amplified product withreal-time qPCR and analyze the signals.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method for detecting a glycosylated target protein in a sample, comprising: (a) contacting a sample comprising a probe-labeled glycosylated target protein with: (i) a first conjugate comprising a first nucleic acid tag linked to a first capture agent that specifically binds the target protein; (ii) a second conjugate comprising a second nucleic acid tag linked to a second capture agent that specifically binds the probe; and (iii) a bridging nucleic acid that hybridizes to the first and second nucleic acid tags; under conditions sufficient to specifically bind the first and second capture agents to the probe-labeled target protein and to hybridize the bridging nucleic acid to the first and second nucleic acid tags to produce a target protein-bound nucleic acid complex; and (b) detecting the target protein-bound nucleic acid complex.
 2. The method of claim 1, wherein the target protein-bound nucleic acid complex comprises an amplicon and the detecting comprises: amplifying the amplicon to generate an amplification product; and detecting the amplification product to provide for detection of the glycosylated target protein.
 3. The method of claim 1, wherein the bridging nucleic acid comprises a first region complementary to the first nucleic acid tag and a second region complementary to the second nucleic acid tag.
 4. The method of claim 1, further comprising, prior to step (a), contacting a sample comprising a metabolically tagged glycosylated protein with a reactive probe to produce the probe-labeled glycosylated target protein.
 5. The method of claim 4, wherein the sample is obtained from a eukaryotic cell comprising the metabolically tagged glycosylated protein.
 6. The method of claim 5, wherein the method further comprises contacting the eukaryotic cell with a tagged sugar under conditions sufficient to produce the metabolically tagged glycosylated protein.
 7. The method of claim 4, wherein the metabolically tagged protein comprises a first chemoselective tag.
 8. The method of claim 7, wherein the first chemoselective tag is an azide.
 9. The method of claim 4, wherein the reactive probe comprises a second chemoselective tag selected from the group consisting of an alkyne, an azide, a phosphine, a thiol, a maleimide or iodoacetyl, an aldehyde, an alkoxyamine.
 10. The method of claim 9, wherein the second chemoselective tag is an alkyne.
 11. The method of claim 1, wherein the first capture agent and the second capture agent are independently selected from a nucleic acid, a protein, a peptide, or a small molecule.
 12. The method of any one of claims 1-11, comprising determining the amount of total target protein in the sample.
 13. The method of claim 12, wherein said determining is carried out using a proximity-based ligation assay comprising: (a) contacting the sample with: (i) a third conjugate comprising a third nucleic acid tag linked to a third capture agent that specifically binds a first epitope in the target protein; (ii) a fourth conjugate comprising a fourth nucleic acid tag linked to a fourth capture agent that specifically a second epitope in the target protein; and (iii) a bridging nucleic acid that hybridizes to the third and fourth nucleic acid tags; under conditions sufficient to specifically bind the third and fourth capture agents to the probe-labeled target protein and to hybridize the bridging nucleic acid to the third and fourth nucleic acid tags to produce a target protein-bound nucleic acid complex; and (b) detecting the target protein-bound nucleic acid complex.
 14. The method of claim 12, comprising comparing the level of glycosylated target protein to the level of total target protein.
 15. A composition, comprising: (a) a first conjugate comprising a first nucleic acid tag linked to a first capture agent that is capable of specifically binding a target protein; and (b) a second conjugate comprising a second nucleic acid tag linked to a second capture agent that is capable of specifically binding a probe.
 16. The composition of claim 15, further comprising: (c) a bridging nucleic acid that is complementary to the first and second nucleic acid tags.
 17. The composition of claim 15, wherein the first capture agent and the second capture agent are independently selected from a nucleic acid, a protein, a peptide, or a small molecule.
 18. The composition of claim 15, wherein the first capture agent is an anti-target protein antibody and the second capture agent is an anti-biotin antibody or an avidin moiety.
 19. The composition of claim 15, wherein the composition further comprises a probe-labeled glycosylated target protein.
 20. A kit, comprising: a first conjugate comprising a first nucleic acid tag linked to a first capture agent that is capable of specifically binding a target protein; and a second conjugate comprising a second nucleic acid tag linked to a second capture agent that is capable of specifically binding a probe.
 21. The kit of claim 20, further comprising: a bridging nucleic acid that is complementary to the first and second nucleic acid tags.
 22. The kit of claim 20, wherein the first capture agent is an anti-target protein antibody and the second capture agent is an anti-biotin antibody or an avidin moiety. 